Techniques

Brightfield

With transmitted light microscopy, images result from light (halogen lamp source) passing through the specimen. Specimen 'details' will be evident if the specimen and background differently alter the phase of light – giving contrast between the specimen and background. Since cells are made mostly of water (70%), they do not alter the passage of light enough to provide much of an image. Therefore, when imaging a specimen that is poorly visible, contrast may be increased by fixing and histologically staining the specimen, or by using microscope optics to accentuate the small differences in the phase of light that the specimen causes (ie DIC, phase contrast, dark-field).

Differential Interference Contrast (DIC)

With DIC microscopy, two slightly separate, plane polarized beams of light are used to create a 3D-like image of the unstained specimen. A polarizer, placed just after the light source, creates plane polarized light that is passed through a Wollaston prism which splits the beam into two beams that have their axis of vibration at 90° relative to each other. The beams cross and go through the condenser and emerge separated by a small distance. The two beams go through slightly different parts of the specimen where small differences in refractive indexes of the cellular components alter their wave path lengths differently. Next, the two beams enter the objective, and pass another Wollaston prism that recombines them. This second prism is offset slightly from the first, creating a difference between deviated and undeviated beams. Since the two beams went through slightly different parts of the specimen, they will have different path lengths; therefore the waves can interfere if they are vibrating in the same plane. Therefore, the combined beam passes a second polarizing filter that is 90 degrees to the first one, which polarizes the bipolar beam and so the combined polarized light can have negative interference – gives light and dark areas of specimen.

Phase Contrast

Phase contrast microscopy uses an annular stop in the condenser and a phase plate within the objective lens, which is aligned with the annular stop. In this configuration, the light path can be split and each of the separated beams will pass through the specimen at the same place, as a cone of light at the focal point. Any background light, which is un-deviated by the specimen, will go through the phase ring in the phase plate and is advanced by a quarter of a wavelength. Deviated light passing through the specimen is retarded by a quarter of a wavelength and passes through the phase plate without going through the ring. When the beams are recombined further along the light path, the differences in the phase of the deviated and un-deviated light beams become additive and subtractive. The resultant wave is the sum of the two waves which have their crests and troughs opposite each other. The difference in amplitude can be seen as a change in brightness, since brightness is proportional to the square of the amplitude. The net result is that features of the object are either lighter or darker than the surrounding field.

Dark-Field

With Dark-Field microscopy, oblique light rays illuminate the specimen, and are not transmitted directly through the objective. Only light rays that are diffracted by the specimen into the objective aperture are collected resulting in a dark background and a bright specimen.

Epifluorescence Microscopy

With epifluorescence microscopy, the entire specimen is flooded evenly in excitation light. All parts of the specimen are excited simultaneously and the resulting fluorescence (emission) is detected by the detector or camera. Epifluorescence microscopy is a powerful tool for visualizing sub-cellular structures, however, is prone to image distortion due to unfocused light (emanating from above and below the plane of focus) reaching the detector.

Optical Sectioning Fluorescence Microscopy

Compared to conventional fluorescence microscopy, out of focus blur, resulting from emission above and below the focal plane, can be overcome by 'optically sectioning' the specimen at the plane of focus. Examples of optical sectioning include: confocal laser scanning microscopy, multi-photon confocal microscopy, stimulated emission depletion microscopy, and structured illumination.

Restorative Deconvolution

A post-acquisition procedure which re-assigns light associated with widefield fluorescence imaging. The distorted signal is mathematically deconvolved using the point spread function. The result is a high quality image with substantially less noise, better definition and higher resolution in 3D.

Structured Illumination – ApoTome

Optical sections can be obtained in epifluorescence microscopy using structured illumination. A plate with an imprinted grid is inserted into the field diaphragm plane in the illumination pathway and projected onto the specimen. The projected grid image is translated over the specimen by tilting the glass plate back and forth. At least three raw images of the specimen are acquired with the grid superimposed in different positions. These images are subsequently processed automatically by the microscope software to create an optical section. The software determines grid contrast as a function of location and removes out-of-focus image information before assembling the three images into a final single image.

Confocal Laser Scanning Microscopy (CLSM)

Photo credits: John Bergin

A major limitation of widefield fluorescence microscopy arises from the fact that the sample is illuminated throughout its volume regardless of the focus plane, and fluorescence is collected not only from the focus plane, but also from planes above and below it. As a result of image distortion by out-of-focus light, widefield fluorescence images often demonstrate compromised specimen detail and contrast. Confocal microscopy largely eliminates out-of-focus haze by incorporating a pinhole near the detector that resides in a plane conjugate with the focal plane, allowing only in-focus-light through to the detector. The result is an image of fluorescence signal arising from the plane of focus, or an optical section at the plane of focus, with out-of-focus light eliminated by the pinhole.

Stimulated Emission Depletion (STED) Microscopy

Stimulated emission depletion (STED) creates super resolution images by altering the effective point spread function of the excitation beam using a second laser, in the shape of a doughnut surrounding the excitation beam, which suppresses fluorescence emission from fluorophores outside the hollow center of the 'doughnut'. Therefore, only fluorophores located in the very center of the 'doughnut' will be excited by the excitation beam. An image is created by raster-scanning the specimen with the excitation/depletion laser combination.

Multi-Photon Confocal Microscopy

Multiphoton (MP) microscopy uses pulsed long-wavelength light as an excitation source. The fluorophores must absorb enough energy for excitation, which requires two photons of long-wavelength light (compared to just one photon of short wavelength). The MP microscope does not use a pinhole to produce optical sections, rather, depends on concentrating the excitation lasers to the plane of focus, where the density of long-wavelength photons will be high enough to achieve 2-photon excitation. Therefore, only fluorophores at the plane of focus will be excited, and emission light detected will be only that of in-focus-light. The long-wavelength, low energy excitation lasers of MP microscopes possess low photo-cytotoxicity, photobleaching, and photodamage effects compared to the short-wavelength, high energy excitation with traditional confocals. As a result, MP microscopy is well suited for live cell imaging, where cells may be observed for longer periods with fewer toxic effects. As well, long-wavelength light can penetrate deeper into the specimen, allowing for the visualization of thick living tissue samples.

Fluorescence Recovery After Photobleaching (FRAP)

For FRAP analysis, a region of interest is defined and is bleached by brief illumination with high laser intensity. Once the region of interest is bleached, the progress of fluorescence recovery is recorded over time. Changes in intensity in the bleached region represent the sum of all movements of the fluorescent molecules, whether passive or active. The regeneration time (half-recovery period) is a measure of the speed of protein movement.

Fluorescence Loss in Photobleaching (FLIP)

For FLIP analysis, a region of interest is defined for bleaching. Brief illumination of the region with high laser intensity repeatedly during the course of imaging ensures it remains bleached. The progress of fluorescence decay in the adjacent non-bleached area is recorded. Changes in intensity in the non-bleached region represent the sum of all movements of the fluorescent molecules, whether passive or active. The decay of fluorescence (half-life) is a measure of the speed of protein movement.

Fluorescence Resonance Energy Transfer (FRET)

FRET analysis enables the quantitative temporal and spatial information about the binding and interaction between proteins, lipids, enzymes, DNA and RNA. FRET is a process in which energy is transferred from a fluorescent donor molecule to a fluorescent acceptor molecule. FRET can be detected as sensitized emission of the acceptor, where emission of an excited donor molecule specifically excites the acceptor molecule, thus increasing its emission. The efficiency of the transferred energy depends on the molecular distance between the two fluorophores. Energy transfer from donor to acceptor depletes or "quenches" the excited state population of the donor, and FRET will, therefore, reduce the fluorescence intensity of the donor. Photobleaching the acceptor to relieve this quenching of the donor (termed "acceptor photobleaching") offers another option for detecting FRET in vivo (Trinkle-Mulcahy et al., 2007).

Photo-activation/Photo-conversion

Credits: Leica Microsystems

Molecular or optical highlighters are fluorophores that can change color or emission intensity as a result of external photon stimulation or the passage of time. Examples of such fluorophores include:

Photo-activatable highlighters: produce no or little fluorescence, but dramatically increase their fluorescence intensity after activation by irradiation at a specific wavelength. For example, unconverted PA-GFP is optimally excited by 405 nm light, and demonstrates negligible absorbance by 488 nm light. After conversion with 405 nm light, PA-GFP absorption optimum is shifted to 504 nm, and will demonstrate an approximate 100-fold increase in green fluorescence by excitation with 488 nm light.

Photo-conversion highlighters: undergo a change in fluorescence emission profile upon irradiation at a specific wavelength. For example, the Dendra2 fluorophore emits light in the green range, but when 'converted' by irradiation in the UV range (ie 405 nm), will emit light in the red range.

Photo-switchable highlighters: have reversible on/off switching capabilities. For example, the Dronpa fluorophores can alternate between a dark state when irradiated with 405 nm light, and a fluorescent state when irradiated with 488 nm light.

*Available on Workstation 1*

Colocalisation

The Colocalisation module can be used to unmix the overlapping signals of different fluorescence channels. To do this, the ZEN software makes use of modern methods, such as the automated threshold value method of Costes et al. Meaningful results on the colocalization of proteins can be obtained as a scatter plot, image and data table. If you only want to analyze certain areas of the image, you can simply mark the corresponding region directly in the image and scatter plot. Spectral Colocalisation for ZEN then supplies the data both for the whole image and the selected regions. Of course you can also export your measured values in CSV format and process them further using a spreadsheet.

Deconvolution

Due to the optical properties of the microscope system, light from the areas above and below the focal plane also enters the image. This means that fine structures on the specimen can no longer be picked out. It is precisely here that the 3D Deconvolution software module starts, by calculating stray light in the image stack back to its place of origin. The distorted specimen structure unfolds and is displayed more sharply. This technique also reduces any image noise that may be present, making a decisive contribution to the first-class image quality of your image stacks and 3D models.

3D VisArt

With 3D VisArt you can display multi-dimensional image stacks as 3D volume models. The ZEN module processes up to 8 different channels and, besides the spatial image coordinates, also takes the time lapse information relating to your images into consideration.

You can position a calculated three-dimensional model interactively according to the view that you require and provide it with annotations, coordinates and scale axes. It goes without saying that 3D VisArt also provides you with powerful tools for the interactive measurement of distances and angles. Would you like to present or publish the result afterwards? If so, simply export the images and animations into common image formats, such as TIF, JPG or AVI.

Image analysis

Would you like to generate automatic measurement procedures simply yourself? The ZEN Image Analysis module makes it possible for you to do this, even without programming knowledge. With our measurement program wizard you tackle even complex measurement tasks yourself in just a few minutes. Once created, the programs are always available to be used for an unlimited number of images. You retain full control over the measurement process at all times.

Each step can be changed interactively before it is performed. Even processes that run automatically can be easily interrupted if necessary, allowing you to adjust the settings to your requirements. If desired, you can of course also use the Image Analysis module to export all your measurement data simply in the Excel-compatible CSV table format and process them further as you wish.

Interactive Measurement

Measure microscopic structures interactively. Generate gray value profiles of cell organelles. Trace the contour of a cell nucleus with your mouse to determine its area. Count cells simply by clicking on them.

With the ZEN and the Interactive Measurement module you define yourself which of the 90 plus morphological parameters available are measured in your images. The Contour tool, for example, allows you to determine the area, diameter and perimeter of a cell simultaneously. You perform a comprehensive and precise analysis of the cell in a single operation, even in the online image. For each tool you can specify which measurement results you want ZEN to show in your image.

*Available on Workstation 1*

Imaris

Imaris is Bitplane’s core scientific software module that delivers all the necessary functionality for data management, visualization, analysis, segmentation and interpretation of 3D and 4D microscopy datasets. Combining speed, precision and ease-of-use, Imaris provides a complete set of features for working with three- and four-dimensional multi-channel images of any size, from a few megabytes to multiple gigabytes in size.

Search, tag, and manage your data
Organize and manage full experiments
Premier Volume Rendering
Spots and Surfaces, Segmentation and Interaction
Create video and montage of 3D microscopy images

Imaris MeasurementPro

Imaris MeasurementPro enables researchers to extract critical statistical parameters from their microscopy images thus allowing for the quantification of scientific findings. Imaris MeasurementPro adds shape, size, and intensity based measurement capabilities to the volume rendering, surface rendering and object detection features of Imaris. It allows researchers to interactively classify, group, and filter segmented objects based on any of the calculated statistics.

Obtain, view and export statistics
Obtain precise measurements of intensity values
Select objects of interest for extracting parameters
Sort and classify objects in real-time based on parameters
Measure distances between points within an object

Imaris Track

ImarisTrack is the most powerful commercially available live cell imaging, tracking & analysis software that rises to the challenge of monitoring temporal changes in biological systems (2D and 3D images over time). ImarisTrack is based on a choice of multiple sophisticated manual and automatic tracking algorithms and also includes an intuitive and interactive track editing tool if manual correction is necessary.

Automatically track objects in 3D + time
Choose from a large variety of tracking algorithms
Handle thousands of objects and time points
Manually edit, create or revise tracks
Generate object and track statistics based on changes

Imaris Coloc

Obtaining accurate information about the position of stained tissue and cellular components is the primary goal of digital microscopy. ImarisColoc has been designed to give researchers the most powerful colocalization analysis tool to quantify and document co-distribution of multiple stained biological components.

Multiple colocalization selection methods
Obtain statistics in real time
Present data as new 3D or 4D color channel
Expand or narrow the computed histogram region
Co-localization of entire time series analyzed in fewer steps

Imaris Cell

ImarisCell is an Imaris module specifically designed for the analysis of 2D, 3D, and 4D images of cells and their components. ImarisCell enables researchers to qualitatively and quantitatively examine micro relationships that exist between cells. ImarisCell also allows researchers to map organelles and other cellular components within individual cells, then examine the relationships between those components.

Examine relationships between cells and cellular components
Utilize biologically meaningful image analysis units
Detect cells based on cytoplasm or plasma membrane labelling
Detect and classify vesicular objects of various populations
Automatically works on 2D, 3D or 4D datasets

Filament Tracer

FilamentTracer is the most advanced software product for the automatic detection of neurons (dendritic trees, axons and spines), microtubules, and other filament-like structures in 2D, 3D and 4D. When combined with ImarisTrack, detection of temporal changes in length and volume of developing spines and dendrites will help researchers understand alterations caused by developmental and environmental changes.

Versatile tracing methods
Obtain branch length and diameter statistics
Manually draw segments with automatic centering
Utilize multiple branch selection methods
Visualize filaments along with non-filamentous structures

Imaris Batch

ImarisBatch is an Imaris subsystem for the processing of 2D, 3D, and 4D image series in batch. This subsystem saves valuable time when running repetitive jobs because processing can be queued and completed without interaction from the user. Since the introduction of Imaris 8.0 batch operations can be triggered directly from the Arena view. The user can execute a pre-defined image analysis protocol on all images of one group or all images from multiple groups. The results of batch analysis can be directly displayed and explored in Imaris Surpass or Imaris Vantage.

Save valuable time by batch processing
Reproduce exact analytical procedures
Decouple time consuming procedures from interactive sessions
Batch process the full experiment and get the results which preserve the experiment structure
Utilize large servers for parallel processing

Imaris Vantage

Welcome to a new era of data interpretation, plotting and presentation. This module enables you to compare and contrast experimental groups by visualizing your image data (segmented and original) in five dimensions as uni- or multi-variate scatterplots. Along with the use of box + whisker (5-number summary) plots Vantage will help you interpret intrinsically complex and dynamic phenomena. ImarisVantage enables researchers to dissect their multi-dimensional, multi-object images by creating a series of fully customizable plots for better understanding of hidden relationships and associations between calculated measurements, objects or groups of objects.

Dissect multi-dimensional, multi-object images images
Visualize your image data in 5 dimensions
Create fast, explorative and interactive multi-dimensional plots
Create side by side ready to publish one dimensional plots to compare groups
Export figures and annotations into your presentations

Imaris XT

ImarisXT is a multi-functional two-way interface from Imaris to classic programming languages, Matlab and other imaging software programs. It allows rapid development and integration of custom algorithms that are specific and tailored to scientific applications where generic image processing would fail.

Develop custom algorithms for image processing and segmentation
Answer challenging questions with targeted tools
Integrate new modules with a customizable user interface
Exchange data with Matlab, Java and Python
Free access to pre-existing library of XTensions

*Full Zen Blue Deconvolution on Workstation 2*

AxioVision Base

Adjust display parameters without manipulation of pixel intensity values
Assign annotations and geometric scalings to acquired images instantly
Store microscope- and camera settings as meta information directly in the ZVI image
Save images in compressed ZVI file format
Specific interface configurations for Windows multi-user environment
Image import: lsm, bmp, tif, jpg, j2k, jp2, gif, tga, png, cal, mac, msp, ras, pct, eps, wmf, psd, img, cmp
Image export (bmp, jpg, j2k, tif, tga, png, psd, img, cmp, avi, mov
Measure intensity profiles like length, area, perimeter, circle or angle interactively
Count and mark events on images
View multidimensional images in every dimension with the gallery view
Compare images in splitter display mode synchronous or independent

3D Deconvolution

The AxioVision devolution module offers adjustable strength 3D deconvolution using theoretical or measured PSF. This module contains four approaches for deconvolution Nearest Neighbour, Regularized Inverse Filter, Fast Iterative and Iterative.

Extended Focus
A microscope’s depth of field alone is often insufficient to obtain an image that is sharp over the entire thickness of the sample. The solution is to simply acquire a number of images whilst focusing through the foreground and background of the specimen. The Extended Focus module then combines the sharp areas of each plane to create an image that is sharp throughout. The Extended Focus module analyzes each individual image in accordance with the innovative wavelet method. Not only is the area surrounding each sharp area analyzed, but the entire image content is also interpreted.

AutoMeasure

If you need to create automatic measurement routines yourself: with the AutoMeasure module you can rapidly obtain precise results without complicated programming. With the help of the measurement wizard you carry out even complicated measurements within a few minutes. Simply define the measurement parameters that you need and you can measure an unlimited number of images – while completely controlling the measuring process. Even automatic processes can be interrupted at any time and all parameters individually adjusted with the interactive function dialog.

Interactive Measurement

Measure microscopic structures interactively. Simply select your desired parameter from more than 90 different options. Our measurement wizard then records all the data in the sequence that you have specified. The results may be archived together with the image or exported as a table.

CBIA techniques

Brightfield

Differential Interference Contrast (DIC)

Phase Contrast

Dark-Field

Epifluorescence Microscopy

Optical Sectioning Fluorescence Microscopy

Restorative Deconvolution

Structured Illumination – ApoTome

Confocal Laser Scanning Microscopy (CLSM)

Stimulated Emission Depletion (STED) Microscopy

Multi-Photon Confocal Microscopy

Fluorescence Recovery After Photobleaching (FRAP)

Fluorescence Loss in Photobleaching (FLIP)

Fluorescence Resonance Energy Transfer (FRET)

Photo-activation/Photo-conversion

Contact us

Cell Biology and Image Acquisition Core Facility

Who to contact?

Techniques

CBIA techniques

Brightfield

Differential Interference Contrast (DIC)

Phase Contrast

Dark-Field

Epifluorescence Microscopy

Optical Sectioning Fluorescence Microscopy

Restorative Deconvolution

Structured Illumination – ApoTome

Confocal Laser Scanning Microscopy (CLSM)

Stimulated Emission Depletion (STED) Microscopy

Multi-Photon Confocal Microscopy

Fluorescence Recovery After Photobleaching (FRAP)

Fluorescence Loss in Photobleaching (FLIP)

Fluorescence Resonance Energy Transfer (FRET)

Photo-activation/Photo-conversion

Live Cell Imaging/Time Lapse arrow_drop_down

Z-Stack arrow_drop_down

Multiple Positions arrow_drop_down

Tile Scanning arrow_drop_down

FIJI (Is Just ImageJ) arrow_drop_down

Zen Black (Desk) arrow_drop_down

Zen Lite arrow_drop_down

Imaris arrow_drop_down

Zen Blue arrow_drop_down

AxioVisionLE arrow_drop_down

AutoQuant X2 arrow_drop_down

Cell Profiler arrow_drop_down

Contact us

Cell Biology and Image Acquisition Core Facility

Who to contact?