BIT Microscopy Final

What parameters can you adjust in the acquisition software of the confocal microscope and what strategies do you use to get the best images? What are the tradeoffs

Examples of adjustable parameters - light path parameters, laser power, detector gain, pinhole, scan speed, scan size etc. What are the tradeoffs ---examples: opening the pinhole lets more light through (increases signal) but increases thickness of the optical section, thereby lowering axial resolution; increasing detector gain above recommended setting increases noise.

What kind of parameters can we quantify when we use software for analysis of microscopic images?

Examples: • Size and Shape • Surface Area • Distance, Length, Perimeter • Density/Intensity • Localization • Dynamic changes of any parameter over time • Particle Tracking, Co-localization, FRAP, FRET, etc.

Light Sheet Fluorescence Microscopy

Illumination occurs from the side and does not go through detection objective. No pinhole, only thin section is illuminated by scanned laser beam and all emission is detected. Detector: Camera (high quantum efficiency) Sample is embedded in a hydrogel and suspended in a chamber. Can be positioned in x,y,z and rotated. It is moved through the sheet of light for 3D acquisition. High speed 4D imaging of transparent organisms over extended time periods (several days) with minimal bleaching. High speed 3D imaging for cleared samples (requires special objectives and chambers). Drawbacks: Only suitable for transparent specimen that fit in the chamber. Data management is challenging. Data sets of 2 TB and more are common. Many systems are only suitable for imaging either cleared samples or samples that are suspended in a saline solution because of refraction issues.

Objective choice for maximum light transmission:

I ~ NA^4/Mag^2 NA Magnification If signal is low use a high NA objective with medium magnification (e.g. 40x water or oil) Do not use phase lenses, use objectives with less lenses (sacrifice correction) Remove analyzer and DIC prism if not needed

Imaris

3D Microscopy Image Analysis Software Multi-channel data visualization (up to 100s of Gigabytes) Automated 3D rendering Multiple file formats recognized Object detection Algorithms appreciated by the community Motion analysis Interaction analysis Machine learning classification of detected objects

Structured Illumination Microscopy (SIM) Pros/Cons

A grid is projected in the light path at different angles to reveal high frequency structures that were not resolved. 2x better resolution than confocal in x,y and z Slow since grid needs to be rotated, only works if grid pattern can be projected into the sample (not good for thicker specimen) Projecting a lattice pattern (lattice SIM) makes this process faster

Automatic segmentation - Thresholding

A type of segmentation which classifies different parts of an image based upon pixel intensity Useful for automatically identifying and separating objects from the background in an image Converts images to binary: - Objects with pixel intensities above a certain threshold are reassigned a value of 1 (all white) - Everything with pixel intensities below the threshold are converted to values of 0 (all black) Sensitive to noise and uneven background - it is very important to generate high quality images with optimal signal/noise ratio

Beam Splitters

AOBS (Acusto Optical Beam Splitter) The Leica confocal models have a tunable Acusto Optical Beam Splitter AOBS instead of dichromatic beam splitters. Prism: in Leica systems Split light in color components and divert to separate detectors with sliders (emission band is adjustable and can be adapted to match your fluorophore). Spectral detector can be used to acquire a whole emission spectrum in one scan Conventional beam splitters Zeiss confocal models have conventional beam splitters arranged in 2 filter wheels. Diffraction grating: in Zeiss systems Use primary and secondary dichroic beamsplitters in conjunction with conventional emission filters (less flexibility) The diffraction grating separates light into colors as the light passes through the many fine slits of the grating. Dichroic filters separate a broad spectrum of light into two components: a reflected component and a transmitted component.

Optimize Scan Parameters for your purpose

Adjust scan speed (pixel dwell time) and/or averaging (line or frame) to improve signal to noise ratio Choose pixel array in x,y; zoom factor Do you need to get an overview, adjust for highest resolution? Do you need resolution or speed? You can zoom in a small area and sample at optimal pixel size. (Often limited by photostability of the fluorophore and phototoxicity.) Gray level resolution (8 bit, 16 bit). Use higher bit rate if you want to quantify the data or if you have a low signal. Opening the pinhole lets more light through (increases signal) but increases thickness of the optical section, thereby lowering axial resolution Increasing detector gain above recommended setting increases noise

Confocal Microscopy

Adjustable pinhole in emission path eliminates light rays from image planes that are not in focus. It is in a conjugate image plane. A very strong light source (laser) needs to be used for illumination A focused laser spot (for excitation) is scanned across the sample by two scanning mirrors. Dichromatic beamsplitter and emission filter have the same role as in Epifluorescence. Photomultipliers (PMTs) serve as detectors The image is created point by point.

Filter types (bandpass, longpass etc). Matching filters to fluorescent markers.

An optical filter is a device that selectively transmits light of different wavelengths. A band pass filter is a filter blocking both longer and shorter wavelengths. Wavelength are cut-off both to the left and to the right of its curve. Long pass and short pass filters are two distinct types of specialized optical filters. Long pass filters transmit electromagnetic radiation with long wavelengths while blocking shorter wavelengths. Long pass filters can be used to pass only precise fluorescence wavelengths while blocking leakage from the excitation lamp. Short pass passes short wavelengths and block longer ones. long pass and short pass filters are often used in conjunction to filter a narrow section of light.

Antifade reagents

Antifade reagents prevent photobleaching (many of them are toxic).

Airyscan Fast Mode

Beamshaper for fast acquisition mode - uses 16 elements of the area detector (instead of the 32 channel detector honeycomb layout) Fast Airyscan mode is 4x faster than regular Airyscan with slight trade off in resolution (1.5 x better than regular confocal)

Advantages of Metal Halide Arc Lamps over Mercury Arc Lamps

Better spectral efficiency, especially in the blue and green range More uniform illumination Extended life span (2000h vs 300h) No alignment necessary

Bit Depth

Bit Depth is a function of Analog to Digital Converter Same original information is divided into smaller increments as bit depth increases. A/D converter divides maximum expected signal into gray levels 2^x , x = bit depth 8 bit = 28 = 256 gray levels 10 bit = 210 = 1024 gray levels 12 bit = 212 = 4096 gray levels 16 bit = 216 = 65536 gray levels *Choose bit depth that is equal or higher than dynamic range of detector.*

Super-resolution methods in fluorescence microscopy

Break the barrier of diffraction limited resolution Point spread function - structures smaller than ~200nm are lost in a blur STED: Stimulated Emission Depletion Point spread function engineering. PALM: Photoactivated Localization Microscopy; STORM: Stochastic Optical Reconstruction Microscopy Localize a few points of light at a time SR-SIM: Structured Illumination Microscopy Uses Illumination pattern to down-modulate non resolvable structure

CCD vs CMOS

CCD Imagers: Performance-critical functions take place outside of the sensor and are independent of the sensor. Clocking, timing Waveform generation Gain and Offset Designers can optimize the electronics for specific applications. CMOS Imagers: Key functions are integrated into the sensor chip and performance characteristics are programmed by the chip manufacturer. Driven by large-volume, consumer-oriented markets. Second generation scientific CMOS sensors combine high QE with fast frame rates and low noise.

CMOS Sensor

CMOS: Complementary Metal Oxide Semiconductor Key functions are integrated into the sensor chip and performance characteristics are programmed by the chip manufacturer. Photoelectrons are converted into voltage by each pixel's photodiode-amplifier pair (faster since conversion happens in parallel instead of serial) More pixels on chip (better sampling for wider range of objectives), provide larger field of view Optimized architecture sCMOS (scientific CMOS) sensors combines high QE with fast frame rates and low noise sCMOS also have correction on a pixel by pixel basis to ensure uniform response New and improved back-illuminated sCMOS cameras have similar performance as EMCCDs. When signal is extremely low EMCCD cameras are still better

Cameleons as calcium indicator

Cameleon is an engineered protein based on variant of green fluorescent protein used to visualize calcium levels in living cells.

CCD Cameras

Charge-coupled devices (CCDs) are silicon-based integrated circuits consisting of a dense matrix of photodiodes. Individual wells (pixels) collect photons Photons are converted to electrons (photoelectrons) Electrons are stored in a potential well and transferred across the chip through registers and output to an amplifier Dynamic range: full well capacity / camera noise Two part sensor, one for collection one for storage. Incoming photons are shifted into storage part. While signal is integrated on light sensitive part, stored charge is read out. Efficient and rapid shift of accumulated charge.

Colocalization

Colocalization of fluorophores in confocal microscopy Two or more of the emission signals can overlap in the final image due to their close proximity within the microscopic structure (fluorescently labeled molecules bind to targets that lie in very close spatial positions). Refers to observation of the spatial overlap between two (or more) different fluorescent labels, each having a separate emission wavelength, to see if the different "targets" are located in the same area of the cell or very near to one another. Cross-Excitation yields false colocalization

Confocal vs widefield microscopes

Confocal microscopy prevents out of focus light from being detected by placing a pinhole aperture between the objective and the detector, through which only in focus light rays can pass. Pinhole rejects out of focus light, producing sharp images. In contrast, widefield microscopes allow out of focus light to pass directly to the detector (more blur). Areas above and below the focal plane get illuminated and contribute noise. Confocal microscopy is especially well suited for examining thick specimens Widefield deconvolution processing has proven to be a powerful tool for imaging specimens requiring extremely low light levels.

Digitization

Conversion of analog, continuous signal into a set of discrete values. (the process in which media is made into a computer-readable form).

Cross-Excitation yields false colocalization

Cross-excitation: a fluorophore is not just excited by wavelength at its peak value, but also by wavelength at certain range around the peak, which can extends into the area used by other fluorophores. When emission spectra of two fluorophores overlaps, emission from one channel will extend to another channel. Unfortunately, many commonly used fluorophore pair have more or less overlapped emission spectra which pose the problem of emission bleeding through in most multi-labeling application.

Dynamic range (Lecture 9 Slide 25)

Dynamic range of cameras Dynamic Range is the difference between the lowest and the highest signal level in an image. Wide dynamic range means capturing the low and high signal intensities present. Dynamic range = Full well capacity / readout noise Full well capacity - number of electrons that can be stored in a pixel well Readout noise - read error due to electrical noise measured in electrons

Understand how to read filter specifications: eg. 480/30x; 505DCLP, 535/40m. Explain the meaning of numbers and letters. LP, SP, DCLP, DCXR, DCXRU

Excitation filter (480/30x): Center wavelength is at 480nm; full bandwidth is 30 [ = +/- 15]. "x" is used to define the filter as an excitation filter. Dichroic beamsplitter (505DCLP) The cut-on wavelength is approximately 505nm for this dichroic longpass filter. Dichroic longpass: under 505 is reflected; over 505 is transmitted. Emission filter (535/40m) Center wavelength here is at 535nm; full bandwidth is 40nm [ = +/- 20]. LP -- indicates a longpass filter which transmits wavelengths longer than the cut-on and blocks shorter wavelengths SP -- indicates a shortpass filter which transmits wavelengths shorter than the cut-on, and blocks longer wavelengths DCLP -- dichroic longpass DCXR -- dichroic long pass, extended reflection DCXRU -- dichroic longpass, extended reflection including the UV

Quenching

Excited state relaxation process results in non-radiative energy loss. Often occurs as a result of oxidizing agents or the presence of heavy metals and salts. Energy can also be transferred to other nearby molecules. Fluorescence quenching refers to any process that decreases the fluorescence intensity of a sample.

FLIP: Fluorescence Loss In Photobleaching

FLIP: Fluorescence Loss In Photobleaching Technique used to examine movement of molecules inside cells and membranes. A cell membrane is typically labeled with a fluorescent dye to allow for observation. A specific area of this labeled section is then bleached several times using the beam of a confocal laser scanning microscope. After each imagining scan, bleaching occurs again. This occurs several times, to ensure that all accessible fluorophores are bleached since unbleached fluorophores are exchanged for bleached fluorophores, causing movement through the cell membrane. The amount of fluorescence from that region is then measured over a period of time to determine the results of the photobleaching on the cell as a whole. FLIP involves the study of how the loss of fluorescence spreads throughout the cell after multiple photobleaching events.

FLIP vs. FRAP

FRAP involves the study of a cell's ability to recover after a single photobleaching event. The area that is actually photobleached is the area of interest. There is a single photobleaching event and a recovery period to observe how well fluorophores move back to the bleached site. FRAP which is primarily useful for determining mobility of proteins in regions local to the photobleaching only. FLIP involves the study of how the loss of fluorescence spreads throughout the cell after multiple photobleaching events. The region of interest is just outside the region that is being photobleached. Multiple photobleaching events occur to prevent the return of unbleached fluorophores to the bleaching region. FLIP can also be used to measure the molecular transfer between regions of a cell regardless of the rate of movement.

FLIM

FRET in combination with FLIM gives more reliable results FILM - Fluorescence life microscopy. Fluorescence life time is the average time a molecule stays in the excited state before emitting a photon. It is an intrinsic property of the molecule and its environment. The measurement is independent of the a change in concentration (fluorescence intensity) and laser intensity. FLIM FRET determines the fluorescence life time of the donor molecule. When FRET occurs the donor fluorescence gets quenched because of interaction with the acceptor and the fluorescence lifetime decreases.

Fast Airyscan vs regular Airyscan vs regular confocal mode:

Fast Airyscan mode is 4x faster than regular Airyscan or confocal mode Fast Airyscan mode has 1.5x better resolution than regular confocal mode Regular Airyscan mode has 1.7x better resolution than regular confocal mode Fast Airyscan mode has 4 times better S/N than regular confocal Regular Airyscan mode has 4-8 times better S/N than regular confocal

Draw and label the 3 Filter cube components and explain the role(s) of each filter. Lecture 5 Slide 20

Fluorescence filter blocks have 3 components: 1) excitation filter 2) dichromatic beamsplitter 3) emission filter The dichromatic beamsplitter reflects the excitation light onto the specimen and prevents it from entering the emission path. The light then impinges upon the excitation filter where selection of the desired band and blockage of unwanted wavelength occurs. The selected wavelengths, after passing through the excitation filter, reach the dichromatic beamsplitting mirror, which is a specialized interference filter that efficiently reflects shorter wavelength light and efficiently passes longer wavelength light. The dichromatic beamsplitter is tilted at a 45-degree angle with respect to the incoming excitation light and reflects this illumination at a 90-degree angle directly through the objective optical system and onto the specimen. Before the emitted fluorescence can reach the eyepiece or detector, it must first pass through the barrier or suppression filter. This filter blocks (suppresses) any residual excitation light and passes the desired longer emission wavelengths.

Signal-to-noise ratio (SNR or S/N)

Image Quality is defined by Signal-to-noise ratio. Signal-to-noise ratio (SNR or S/N) compares the level of a desired signal to the level of background noise. Signal needs to accumulate faster than noise.

FRAP and FLIP

Fluorescent molecules lose their ability to emit light and are referred to as bleached or photobleached FRAP and FLIP are used to measure the ability of a molecule to move around over time. To do this, a fluorophore must be covalently attached to the molecule you want to study (i.e. protein, lipid, carbohydrate)

BiFC (bimolecular fluorescence complementation)

Fluorescent protein fragments (not fluorescent) are attached to proteins that are postulated to interact and expressed in live cells. When protein-protein interaction occurs the two complementary fluorescent protein parts get close together, unfold and reform in its native three-dimensional structure.

Fluorograms for analyzing co-localization

Fluorograms are an objective measure of co-localization Images must be of thin sections Region to analyze must be carefully chosen Dyes, filters & excitation must be carefully chosen to avoid cross talk

Quantum Efficiency:

Fraction of photons hitting the detector that generate a response; Wavelength dependent Quantum efficiency (QE) is the measure of the effectiveness of an imaging device to convert incident photons into electrons.

Gamma adjustment

Gamma is the relationship between the detected gray level (input value) and the gray level that is rendered in the final image (output value) as displayed on the computer screen or in a digital print

Electron multiplying CCD Cameras

Great reduction in read out noise (<4 electrons rms), high Quantum Efficiency (QE) (>90%) Quantum efficiency (QE) is the percentage of incident photons that will release photoelectrons in the sensor, i.e. be detected. High sensitivity (ideal for very low signals), lower dynamic range (not good if sample has areas of low and high signals) Electron multiplying CCD sensors amplify captured signal before it is read out. High voltage in extended multiplication register initiates and sustains an impact-ionization process: Photon-generated charge is amplified before it reaches the on-chip amplifier.

Segmentation

Image segmentation is the process of partitioning a digital image into multiple segments (sets of pixels, also known as image objects). The goal of segmentation is to simplify and/or change the representation of an image into something that is more meaningful and easier to analyze. Segmentation - isolating signals from background

ImageJ

ImageJ is a free, open source image processing program for multidimensional image data with a focus on scientific imaging Standard image processing functions Range of different image formats Programmable with macros Written in Java - multi platform (Win, Mac, Linux) Extensible through "plug-ins" - it is possible to accomplish a wide variety of tasks without having to spend a lot of money for commercially available software Support for different programming languages Fiji is a distribution of ImageJ with many plugins useful for scientific image analysis in fields such as life sciences (Fiji is Just ImageJ)

Fluorescence recovery after photobleaching (FRAP)

In FRAP, a specific area of a cell or tissue is photobleached by intense laser light, removing fluorescence from this area. This area is typically a cell membrane or area where diffusion occurs, such as the nucleus, as FRAP requires fluorescent molecules to move around freely in order to function. Fluorescence in the bleached area will slowly recover as bleached fluorophores move out and healthy fluorophores from other areas move in, hence the name fluorescence recovery after photobleaching. Looks at how much time it takes to recover. Some percentage of molecules don't recover (they do not move away or diffuse away)

Integration Time

In confocal microscopy slowing down the scan increases integration time

Zeiss Airyscan pinhole Explained

In theory, the maximum resolution of a traditional confocal imaging system would be achieved with a pinhole diameter of 0.2 AU, which should result in an ∼1.4× increase in spatial resolution compared with that obtained with the traditional 1-AU pinhole. However, the downside to using a pinhole with a diameter less than 1 AU is the dramatic decrease in signal reaching the detector (95% loss at 0.2 AU). If the signal reaching the detector is decreased, the resulting image quality will also decrease.

How to increase S/N Ratio

Increase Signal: Microscope - Use bright objectives (high NA, medium magnification). - Use objectives with fewer lenses; use filters with high light transmission Detector - Higher QE - Signal Integration - Temporal - longer exposure, higher pixel dwell time - Spatial - pixel binning in camera - Larger camera detector - Signal Intensification Decrease Noise: Lower read noise by binning (CCD cameras), slowing readout rate and by using on chip multiplication gain (EMCCD cameras) Lower dark noise by cooling: 50% reduction per 8°C temperature reduction

Important factors that contribute to the intensity of emission light hitting the detector

Intensity of light source High extinction coefficient of fluorophore High quantum yield of fluorophore High photo stability (low quenching and photobleaching) High light transmission of filters and lenses in microscope Match spectral properties of filter sets to dyes Avoid refractive index mismatch

Histogram stretching

Involves modifying the brightness (intensity) values of pixels in the image according to some mapping function that specifies an output pixel brightness value for each input pixel brightness value. is a simple image enhancement technique that attempts to improve the contrast in an image by 'stretching' the range of intensity values it contains to span a desired range of values

Photobleaching

Irreversible decomposition of the fluorescent molecules in the excited state because of their interaction with molecular oxygen before emission. Permanently is unable to fluoresce

bit (binary digit)

Is the basic unit of information in computing. It is used for storing information. Image Depth determines the dynamic range of digital image Digital images: more bits = more information Bits - range of colors used to display light intensities 1-bit = "0 or 1" = 2 values 2-bit = 22 = 4 values 8-bit = 28 = 256 values 8-bit image has 256 grayscale values: 0 = no light, black 255 = highest light intensity, white 16-bit = 216 = 65536 values

Light source: LASER

Light Amplification by Stimulated Emission of Radiation Laser light is polarized! You do not need the analyzer in the light path to capture simultaneous DIC images of the transmitted light.

Multiphoton Microscopy

Light source: pulsed infra red laser (very expensive!) scanning mirrors, point by point acquisition Detector: mostly none-descanned PMT (photomultiplier) No pinhole necessary; Only in-focus plane gets illuminated; Advantages: IR light is less toxic, less scattering and penetrates deeper; allows deep imaging of thicker specimen. Clearer imaging in deeper tissue layers. Resolution is still dictated by diffraction (no super-resolution method). Drawbacks: Thin samples get "cooked" Need to work in dark room Very expensive

Oil immersion vs water immersion objectives

Oil immersion objectives have limited axial range of resolution and illumination intensities. They perform best with thin specimen that are close to the cover slip. At deeper levels effective resolution is reduced. Use of high-quality oil! Wrong oil can leave film on objective lens. Use always the same formulation as recommended by the manufacturer. Water immersion objectives eliminate spherical aberrations in thicker specimen mounted in aqueous media and perform closer to the theoretical resolution of the objective at greater depth.

Deconvolution

Mathematical approach to restore image detail Image is changed by the optical system Once we know how the optical system changes the image we can calculate what it was like before. Images are taken at defined focus levels and compared to a known or guessed profile of how the optics reproduce images. Different algorithms have been developed. If you use deconvolution you ideally want to sample 2x higher than Nyquist (pixel needs to be 4.6 times smaller than the resolution limit). You can also use deconvolution to further improve resolution and contrast in confocal images. This process digitally removes out-of-focus light and reassigns it to its source, creating a much sharper 3-D image. Nyquist's theorem states that a periodic signal must be sampled at more than twice the highest frequency component of the signal. Image deconvolution is a computational procedure that partially corrects images for the influence of the point spread function of the microscope. The recorded image is actually the convolution of the actual intensity distribution in the object with the point spread function of the microscope. Deconvolution is a computational method that treats the image as an estimate of the true specimen intensity and using an expression for the point spread function performs the mathematical inverse of the imaging process to obtain an improved estimate of the image intensity. It does not influence image resolution but often improves the contrast and crispness of image structures, while decreasing image noise.

Fluorescence Images of Thick Specimen Lack Detail How do we make blurry images sharp?

Mathematical method: Deconvolution Optical method: Confocal microscopy

Precautions you have to take when using arc lamps Mercury Arc Lamps

Mercury Arc Lamps: Mercury arc lamps are most commonly used. Mercury arc lamps can bust circuit boards when they ignite! They can also explode. Make sure the microscope, computer and camera are unplugged before you ignite the mercury lamp (prevents destruction of circuit boards). Leave the mercury lamp on for at least 30 min before switching off (prolongs life of the bulb). When warm from previous session wait until lamp house is cold before switching on again (prevents explosion of bulb and mercury exposure).

Multiphoton Microscopy: How it Works

Multiphoton excitation requires the fluorophore to simultaneously absorb two or more photons. This process only occurs in a region of high photon density (the focal plane). No pinhole needed because fluorescence is only emitted from the focal plane. The concept of two-photon excitation is based on the idea that two photons, of comparably lower photon energy than needed for one photon excitation, can also excite a fluorophore in one quantum event. Each photon carries approximately half the energy necessary to excite the molecule. Excitation results in the subsequent emission of a fluorescence photon with the same quantum yield that would result from conventional single-photon absorption. The probability of the near-simultaneous absorption of two photons is extremely low. Therefore, a high peak flux of excitation photons is typically required, usually generated by femtosecond pulsed laser. The purpose of employing the two-photon effect is that the axial spread of the point spread function is substantially lower than for single-photon excitation.

What has pinholes?

Multiple Pinholes: Spinning Disk Confocal No Pinhole: TIRF: Total Internal Reflection Fluorescence Multiphoton Microscopy Lightsheet Microscopy

Light-emitting diode (LED)

New high-power LEDs are now more often used as an alternative illumination source in fluorescence microscopy. The diverse spectra of LEDs can be matched to the absorption properties of the fluorophores. Compared to arc lamps LEDs are cooler, smaller, and provide a more convenient mechanism to cycle the source on and off, and to rapidly select specific wavelengths.

FRET: Fluorescence (Förster) Resonance Energy Transfer

Non radiative energy transfer from donor to acceptor molecule Emission spectrum of donor molecule must overlap with absorbance spectrum of the acceptor molecule Donor and acceptor molecule need to be close together (1-10nm) Transfer of excitation from a DONOR (D) molecule to an ACCEPTOR (A) without emission of a photon FRET allows visualization of protein-protein interactions which is not possible, even when using High Resolution systems.

Nyquist criterion

Nyquist criterion requires that the sampling frequency be at least twice the highest frequency contained in the signal, or information about the signal will be lost. The Nyquist criterion requires a sampling interval equal to twice the highest spatial frequency of the specimen to accurately preserve the spatial resolution in the resulting digital image.

Other image analysis software packages

Other image analysis software packages Zeiss Zen Black and Zen Blue Arivis 4D 2 Imaris 9.7 Metamorph 7.7 Slidebook 6.0

Optical highlighters: Photoactivation and photoconversion reaction

PA-GFP: no initial fluorescence at normal excitation wavelength for GFP imaging. Fluorescence increases dramatically when activated at shorter wavelength (405nm or 411nm) Kaede: fluorescent protein from a stony coral (Trachyphyllia geoffroyi). Undergoes a UV-induced green to red photoconversion. The fluorescence emission profile changes upon UV-induced cleavage in the chromophore.

PALM

PALM: Photoactivated Localization Microscopy Activate only a few fluorescently labeled molecules and determine their location. Then, either turn them off or wait for them to photobleach . Use another weak pulse of light to turn on a few more, and repeat until you've localized them all.

Photomultipliers

Photomultipliers acquire light through a photocathode (glass or quartz window that covers a photosensitive surface), which then releases electrons that are multiplied by electrodes known as metal channel dynodes. At the end of the dynode chain is an anode or collection electrode. Signal amplification is user controlled by adjusting voltage gain. This needs to be set within a defined range that depends on manufacturer. Too much gain amplifies too much noise. A photomultiplier tube is useful for light detection of very weak signals

Fluorescent Proteins

Produced by some marine organisms naturally (jelly fish, corals), other cells can be transformed to produce the protein. Aequorea victoria and Discosoma (Ds red). No need to permeabilize, fix or microinject cells Can be targeted to specific organelles and attached to other proteins to track their location and study their function Dynamic live cell imaging of cellular structures is possible Often bright and less prone to bleaching A large variety of colors available through mutations. GFP mutations that improved fluorescence intensity, photo stability and spectral characteristics mRFP, expanding color spectrum of both GFP and RFP Pioneer of calcium imaging (developed calmodulin based calcium indicators) GFP based pH indicators

Quantitative FRET Analysis

Quantitative FRET Analysis using Acceptor Bleach Principle: Some donor (CFP) signal is transferred (FRET) to the acceptor (YFP) The acceptor is bleached (chemically destroyed) The donor signal increases (up to 30%) since no energy transfer to the acceptor is possible.

Quantization

Quantization is the digitization of the amplitudes (intensity) The transition between continuous values of the image function and its digital equivalent is called quantization. Quantization is the process of mapping input values from a large set (often a continuous set) to output values in a (countable) smaller set. Spatial and intensity quantization produces digital representation of an analog (continuous) image of an object. The elements of this representation (pixels) are arranged in a table (2D in this example) in which each pixel is described by its coordinates and intensity (value).

STED

STED: Stimulated Emission Depletion Fluorescence excitation is narrowed down in space by simultaneously applying a second spot of light for molecular de-excitation (doughnut-shaped). De-excitation (STED) beam confines molecules to the ground state. Only molecules in the center of the doughnut get excited, leading to narrower airy disk. 12 fold higher lateral resolution compared to confocal microscope, no improvement in axial resolution.

Sample preparation

Sample preparation: Choose correct staining conditions; prepare lots of controls fluorophore choice (absorption efficiency, quantum yield, bleaching, absorption and emission spectra, cross excitation, emission bleed through) Needs to match the capabilities of your microscope and detector Garbage in = garbage out

Sampling

Sampling is the digitization of the coordinates (2D or 3D) The sampling rate determines the spatial resolution of the digitized image, while the quantization level determines the number of grey levels in the digitized image. Continuous optical image of an object is sampled in space (in 2D or 3D) and then intensity (brightness) measured in the sampled points is represented in discrete scale.

Signal in Image Equation

Signal in Image = I x QE x T S: Signal (electrons) I: Input light level hitting detector (photons/sec) QE: Quantum Efficiency of detector (electron/photon) T: Integration Time (sec)

What is Fluorescence?

Some molecules (called fluorophores) can absorb light at a particular wavelength and then re-emit light at a longer wavelength. Requires outside source of energy (light) The absorption of a light photon excites an electron to a higher energy state. When the electron falls back to the ground state a photon is emitted. Fluorescence can be cyclical, with the same fluorophore absorbing and emitting light repeatedly

Applications in fluorescence microscopy:

Specific binding of fluorescently labeled molecules to certain cell organelles and structures (nuclei, mitochondria, membranes) Immunofluorescence Fluorescent proteins for dynamic live cell imaging Biosensors and highlighter fluorescent proteins FRAP: Fluorescence Recovery After Photobleaching FLIP: Fluorescence Loss In Photobleaching FRET: Förster (Fluorescence) Resonance Energy Transfer Co-localization

Spinning Disk Confocal

Spinning Disk with multiple pinholes (20000 and more) Detector: Camera (high quantum efficiency) 30 degrees rotation = 1 image 12 images per disk rotation Higher speed, image is acquired using many pinholes at once. Less bleaching; great for live cell imaging. Also cheaper, unless fully loaded. Drawbacks: Pinhole diameter not adjustable to suit objective NA. Less flexibility to get optimal xyz resolution. Thick, scattering samples will lead to crosstalk between neighboring pinholes - higher background Fluorescence emissions originating from remote focal planes and out-of-focus scattering pass through adjacent pinholes, increasing the background signal haze that obscures the image.

Histogram

Step one - Change the way the images look on screen by adjusting histogram. Histogram tells how many pixels of given intensities are in an image. Histogram adjustments alter the way an image is displayed by altering scaling - without changing pixel values. Gamma - the relationship between the brightness of a pixel as it appears on the screen, and the numerical value of that pixel.

Storm

Storm: Stochastic Optical Reconstruction Microscopy Molecules are labeled with fluorescent probes, and a small percentage of labeled molecules gets excited at a time so that each fluorescing molecule can be seen separately. This allows the molecules to be localized individually. The process is repeated many times, capturing a different subset of molecules with each iteration. A final compilation of the images shows each molecule in its precise location in the cell with nanometer accuracy. Very similar to PALM (multiple different labs came up with similar techniques around the same time)

SIM

Structured Illumination Microscopy (SIM) How it works: Use dark bands (moire fringes) productively: put the information back where it belongs moiré fringes are large-scale interference patterns that can be produced when an opaque ruled pattern with transparent gaps is overlaid on another similar pattern. Performance: 2x Resolution Improvement (100 nm) Requires: Patterned Illumination Laser sources The interaction of the optical line patterns creates a pattern of dark and light bands, the Moiré pattern, superimposed onto the lines. Moiré fringes created by interference of grid structure with sample structure contain high frequency information that is transformed down to low frequencies that can be resolved. This transformation can be computed and yields a resolution enhancement in image space. You need to rotate the grid at different angles.

TIRF Microscopy: How it works

TIR can occur at the glass-water interface. Evanescent field extending ~ 150 nm into the water (wider angle creates thinner field). Fluorescence only from molecules in a thin slice near the glass surface Prism type vs. Objective type (special high NA objective required)

Live Cell Imaging: Keeping your cells happy

Temperature ~37°C pH ~7.2-7.4 5-7 % CO2 Nutrients pH buffer Osmolarity

Fluorescence lifetime

The fluorescence lifetime is the characteristic time that a molecule remains in its excited state before returning to the ground state.

Pinhole

The pinhole is in a conjugate image plane and typically set to the size of the center of the PSF (Airy disk) and measured in Airy units. Pinhole rejects out of focus light and produces a sharp image of the focal plane.

Point Spread Function

The point spread function (PSF) is the three-dimensional diffraction pattern generated by an ideal point source of light. The point spread function is based on an infinitely small point source of light originating in the specimen (object) space. Because the microscope imaging system collects only a fraction of the light emitted by this point, it cannot focus the light into a perfect three-dimensional image of the point. Instead, the point appears widened and spread into a three-dimensional diffraction pattern.

Stokes shift

The wavelength difference between the absorption and emission peaks. Fluorophores with a large Stokes shift can be distinguished from the excitation light more easily

Emission bleed through (cross talk)

This happens when the emission spectra of the fluorescent dyes overlap (red area) in your collection window. In this case you will collect some signal from the first emission peak in the yellow emission window. This is particular annoying when the emission of your second dye is lower. You can avoid emission bleed through and cross excitation by using sequential scanning or choosing fluorophores with well separated spectra Bleed-through occurs when you can see fluorescence from a neighboring channel in the channel of interest. This can occur in experiments that involve labeling with more than one fluorophore at a time.

Point spread function shape

To describe the point spread function in three dimensions, it is common to apply a coordinate system of three axes (x, y, and z) where x and y are parallel to the focal plane of the specimen and z is parallel to the optical axis of the microscope. In this case, the point spread function appears as a set of concentric rings in the x-y plane, and resembles an hourglass in the x-z and y-z planes. An x-y slice through the center of the widefield point spread function reveals a set of concentric rings: the so-called Airy disk that is commonly referenced in texts on classical optical microscopy. Depending upon the imaging mode being utilized (widefield, confocal, transmitted light), the point spread function has a different and unique shape and contour. In a widefield fluorescence microscope, the shape of the point spread function resembles that of an oblong "football" of light surrounded by a flare of widening rings.

TIRF Microscopy:

Total Internal Reflection Fluorescence (TIRF) Fluorescence only from molecules in a thin slice near the glass surface Light Source: Laser Prism or high NA objective to achieve wide enough angle Detector: Camera (high quantum efficiency) High contrast, high axial resolution (300nm) Low photo toxicity (only evanescent field is illuminated) Drawbacks: Only objects that are within 300 nm of the coverslip can be imaged, light scattering can cause problems

Microscopy imaging cameras for different microscope types

Widefield Fluorescnce Microscopy - sCMOS cameras Confocal Fluorescence Microscopy - sCMOS (wider fields of view) and/or EMCCD (highest sensitivity) Super Resolution - sCMOS (wider fields of view) and/or EMCCD (highest sensitivity)

Confocal workflow

Workflow: light path and experimental setup Select laser line for excitation Select dichroic beamsplitter Select emission range When using multiple dyes: Simultaneous (one track) or sequential scanning (multiple tracks)? Check for cross excitation and emission bleed through Use sequential scanning for fixed samples if spectra overlap If you want to image moving structures with multiple dyes simultaneously, choose fluorophores with minimal overlap in absorption and emission spectra Line or frame switching?

Precautions you have to take when using arc lamps Metal Halide Arc Lamps

Xenon arc lamps have lower peak intensities, but they are better at longer wavelengths. Need 2-5 min to reach operating temperature Need to be left on for at least 30 min before switching off Need to cool down before switching on again

Zeiss Airyscan

Zeiss Airyscan technology achieves 1.7 fold higher resolution in x,y,z (after deconvolution) than regular confocal mode. Increases S/R (signal-to-noise ratio) 4-8 fold. Works with all common fluorophores Pinhole is set to 1.25 AU and emission light is projected to a 32 channel detector (honeycomb layout). Airyscan detector contains a hexagonally packed detector array instead of a physical confocal pinhole aperture and unitary detector. Each part of the detector array captures a defined part of the psf. Projects 1.25 AU onto the detector (via zoom optics), where each detector element behaves as a small, 0.2-AU pinhole, while the collection efficiency of a 1.25-AU pinhole is maintained. Moreover, because only 1.25 AU is projected onto the Airyscan detector, the optical-sectioning ability of LSM is also maintained. To extend the resolution beyond what a 0.2-AU pinhole provides, Airyscan uses a linear deconvolution, resulting in a 1.7× increase in resolution in all three spatial dimensions

Zoom

Zooming changes the angle of the scan; scan size decreases and bleaches faster because same laser power is spread over smaller area. Zooming provides real magnification until the Nyquist criterion is fulfilled

Absorption efficiency

describes likelihood of absorption (molar extinction coefficient). Larger extinction coefficient means absorption of a photon is more likely. Extinction coefficient refers to several different measures of the absorption of light in a medium

Dark noise

generated by sensor without signal, heat dependent, accumulates over time and high when long exposure is needed, can be reduced by cooling of detector. Very low in modern scientific cameras. Dark current is caused by thermally generated electrons.

Readout noise

generated during readout process, combined electrical noise from system (converting CCD charge into voltage, A/D processing and conversion), dominant in low light, increases with readout speed. refers to the uncertainty introduced during the process of quantifying the electronic signal on the CCD.

Quantum yield

is the ratio of the number of quanta emitted compared to those absorbed. Between 0.1 and 1 Energy gets lost as heat or photochemical reaction

Signal (Shot) Noise

uncertainty in the counting of photons, is intrinsic to photon statistics of a given image and cannot be reduced. Dominates in high light situations. About 10% of signal.

Main scan modes

x,y,z for collecting z-series x,y,t for collecting time series x,y,z,t for collecting z-series at defined time intervals x,y,λ to gain spectral information about the specimen Spectral scanning with 32 channel pmt array + 2 flanking channels Tile scanning, for larger fields, moves stage in defined steps Spectral scanning is an advanced fluorescent imaging technique that records a series of individual images within a user-defined wavelength range with each image detected at a specific emission wavelength.