Advanced Light Microscopy and Image Analysis Core

Multi-photon Excitation

Multi-photon excitation of GFP imaging over time: Time-lapse movie of mitosis in HT 1080 cells with GFP tagged to Histone H2B. Eight image planes were taken every 15 seconds. Each time-point in the movie is a maximum intensity projection of the volume for that time point. Multi-photon excitation produces images with the lowest possible photon dose, while providing exceptional image detail.
Click here to view a quicktime video.

Multi-photon excitation in live tissue (~325µM thick) Z series volume of rat spinal motoneurons innervating soleus muscle (red) and gastrocnemius muscle (green) retrogradely labeled by intramuscular injection with cholera toxin subunit B conjugated to Alexa 594 and FITC, respectively.
Click here to view a quicktime video.

Bright-Field and SHG

Comparison of bright field and second harmonic generation (SHG) imaging of isolated collagen fibers and mouse skin.

SHG is a nonlinear optical effect. Contrast is generated from variations in a specimen’s ability to generate second harmonic light from the incident light beam, usually a Ti-sapphire LASER.

SHG requires intense laser light passing through a material containing a noncentrosymmetric molecular structure. In order to generate contrast via SHG the material must have specific molecular orientation such that the incident light is frequency doubled, examples of biological materials which are polarizable, and assemble into fairly ordered, large noncentrosymmetric structures are collagen, microtubules, and muscle myosin.
Click here to view a quicktime video.

Wide field Vs. Confocal microscopy

Top - A neonatal rat pyramidal neuron filled with Lucifer Yellow imaged on the BioRad MRC600™ confocal microscope using a 20X oil objective, NA=0.8. Image size is 320 x 425 x 180 µm.
Bottom - The same neuron imaged using a fluorescent microscope.

Confocal Image

A neonatal rat pyramidal neuron filled with Lucifer Yellow imaged on the BioRad MRC600™ confocal microscope using a 20X oil objective, NA=0.8. Image size is 320 x 425 x 120 µm.

Polarized Light Image

True-color polarized light image of different crystals. Top left-glucose, top right-EDTA, bottom left-sodium chloride, bottom right-sodium phosphate) Polarized light microscopy can produce both qualitative and quantitative measurement from birefringent specimens (decomposition of a ray of light into two rays when passing through certain samples). These specimens can be alive, fixed or inanimate. The colors in the image are a result of the interference between light waves passing as they pass through the specimen.

Click on the image to see individual high resolution version images.

True-Color Image

Color image of a histological section with blood vessels clearly visible. Annotations, measurements, and micron bar were added with the Spot™ software package.

Triple-labeled vascular casting of rat cerebral cortex.

This maximum intensity projection of a 3D confocal microscope image stack displays cell nuclei in green, blood vessels in red, and (neuronal) Nissl substance in blue. Image provide by of Chris Bjornsson of the Wadsworth Center

Montaging & Sorting Image

a) A montage of 12 3D image fields recorded along the hippocampal pyramidal layer of a rat brain stained with acriflavine (a nuclear stain). Each field was collected on the BioRad™ confocal microscope using a 40X oil objective, NA=1.0. Image size for each field was 215 x 215 x 60µm.
b) The montage region with each nucleus replaced with a sphere of equal volume and color coded depending on its size.

Triple labeled PtK cells

Maximum intensity projection in X/Y axis (62 slices,0.2µM step size) of a PtK cell stained for microtubules (green), and DNA (red). The images were collect using a 60X objective (1.4NA) on a Nikon TE2000 microscope equipped with filter wheels used for multi-dimensional imaging.

Benefit of deconvolution

Maximum intensity projection in X/Y axis (62 slices,0.2µM step size) of a PtK cell stained for microtubules (green), and DNA (red). The images were collect using a 60X objective (1.4NA) on a Nikon TE2000 microscope equipped with filter wheels used for multi-dimensional imaging. Image on left is "raw". Image on right is deconvolved.
View the 3D rotational QuickTime movie here - Size 22.9 MB.

Fluorescent images of a HT 1080 cell in Prophase stage of mitosis (GFP tagged to Histone H2B). Image on the left is a maximum intensity projection of the volume for that time point (six image planes). Image on right is a maximum intensity projection of the deconvolved volume for that same time point.  See a QuickTime movie of the complete mitosis (images taken every 45 seconds) - Size 2.24 MB.
(QuickTime -- Download the free player)

Fixed Neuron

A multi-wavelength, three dimensional, wide-field immunofluorescence image of a fixed neuron. The projection was generated using an extended depth of field algorithm. Cell body labeled for tubulin is shown in blue, F-actin in green, and presynaptic protein in Red. Specimen courtesy of Natalie Dowell-Mesfin BMS-PhD student

2D Tracing
An image of isolated rat 18 day embryonic hippocampal neurons cultured for 7 days on smooth silicon surfaces. The neuronal processes were labeled for β_III-tubulin , imaged and then traced with the 2D tracing software. The yellow areas (lettered) are the cell soma and the green are the traced processes. The image was collected on the Olympus BX41WI epi-fluorescent microscope with an Optronics Magnafire CCD camera using a 20X NA=0.4 objective lens (field size = 577 x 462 µm).

Multi-Channel Imaging Over Time
Pseudo colored time-lapse movie of MCF-7(green) and 10A (Purple) cells co-cultured. The 10A were imaged by phase microscopy while the MCF-7 were imaged in fluorescence (stably transfected w/GFP). 10 image planes of each channel were taken every 10 minutes for 36 hours. Each time-point in the movie is a maximum intensity or average projection of the GFP or phase volume.
Courtesy of Dr. Spink, Wadsworth Center, Albany NY.
Click Here to See time-lapse movie (20.6 MB .avi)

GFP imaging over time
Time-lapse movie of mitosis in HT 1080 cells with GFP tagged to Histone H2B. Six image planes of were taken every 45 seconds. Each time-point in the movie is a maximum intensity projection of the volume for that time point.
Click Here to See time-lapse movie (5.06 MB .avi)

Optical Projection Tomography (OPT)
The Images are projections from a Z series, at the zero angle position, from a complete 360o rotational series. Left, is multimode image; transmitted light and fluorescent (GFP) of a drosophila larva. The image of the right is the head and partial thorax of a deer fly.

View the 3D Rotational movie of deer fly here - Size 7.94 MB (.avi)
View the 3D Rotational movie of a drosophila larva here - Size 4.51 MB (.avi)

Macro Imaging
Image of a small gear, demonstrating both annotation and measurement capabilities of the software.
Courtesy of Mr. Steve Meyer, Wadsworth Center, Albany NY.

Macro Imaging
Filter and tobacco from suspect cigarettes.
Courtesy of Dr. Webber, Wadsworth Center, Albany, NY

Macro Imaging
Freshwater shrimp from water supply.
Courtesy of Dr. Braun-Howland, Wadsworth Center, Albany, NY

FRAP
Photo bleached spot in a lipid bi-layer and the subsequent diffusion into the bleached area. The recovery after bleaching demonstrates the fluid nature of the bi-layer. (Bar=30µM, Time in minutes)
Courtesy of Dr. Kane, RPI, Troy, NY

Opaque Objects
Pseudo colored crystal of mycoplasma arthritidis mitogen.
Courtesy of Dr.Li, Wadsworth Center, Albany, NY

Benefit of Deconvolution Bright Field Imaging
Neuron imaged in bright field mode (left) and the same neuron after deconvolution (right).
Courtesy of Dr. Synder, Wadsworth Center, Albany, NY