Announcements/Outline

• Handout: Fluoview 300 manual (contents)

• Start up your computers for a Java Tutorial today.

• Paper for discussion next time: Robb and Alvarado (2002) – PDF available on web site.– Come prepared to

participate• Lecture topic next time:

Digital Imaging• Dinner at Dr. Hertzler’s?

1. Crossover revisited

2. Reflection imaging

3. TBA: Reflection imaging and Sequential Scanning

Midterm Exam Results

• Average = 67/99 (68%)

• High = 78/99 (79%), 2 students

• Curved grades (90, 80, 70% of 78/99):– A (70-78): 5– B (62-69): 4– C (54-61): 3

• Answer key will be posted in hall beside BR 179

Confirmation Questions

17. The blue argon laser produces what wavelength?

18. The green helium neon laser produces what wavelength?

A. 488 nm

B. 514 nm

C. 543 nm

D. 568 nm

E. 633 nm

Posterization solutions

• Click the <LUT> button to display the Color Tool, and confirm that Intensity Mapping Low = 0, High = 4095, Gamma = 1.

• Save as 24-bit TIF files instead of 8-bit TIF.

• Save to the 2nd imaging computer.

• You can save every image in TILEd displays by selecting “all images” during save display.

Comments on Reports and Project Proposals

• Probes for antibody labeling: Mouse anti-tubulin or anti-engrailed primary antibodies, goat anti-mouse IgG secondary antibody, conjugated to AlexaFluor 546.

• Reflection artifacts in CH1 from BPAE cells are caused by not having the filters in on the scan head.

• Use Figure Legend format to describe images.

• Tardigrades, Daphnia, and planarians are ordered and should come in this week.

• Fertile chicken eggs have been ordered from two local farms.• 22610 neuronal antibody has been ordered. Rhodamine-phalloidin

and AlexaFluor 546-goat anti-mouse antibodies are available from me anytime.

• Rule of thumb for projects: spend about 6-8 hours per week. Keep track of your time, along with what you do, in your lab book.

(a-c) AlexaFluor 488 and Cy3 simultaneous scanning: required for live samples(d-f) AlexaFluor 488 and Cy3 sequential scanning: possible w/ fixed samples

Minimizing crossover: specimen labeling precautions (Molecular Expressions)

1. Choose fluorochromes with as widely separated spectra as possible.

2. Adjust concentrations of fluorescent stains so that intensities are close to equal

3. When selecting fluorescent probes for multiply-labeled specimens, the brightest and most photostable fluorophores should be reserved for the least abundant cellular targets.

Java Tutorial: Crossover compensation

• http://www.olympusconfocal.com/theory/bleedthrough.html• Java tutorial:

http://www.olympusconfocal.com/java/crossoversimulator/index.html– Start with the Swiss Mouse Embryo Cell Culture

Balancing emission intensities reduces much crossover

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Minimizing crossover: instrumental approaches (Molecular Expressions)

1. Absorption spectra are generally skewed towards shorter wavelengths whereas emission spectra are skewed towards longer wavelengths.

• For this reason, multicolor fluorescence imaging should be conducted with the reddest (longest wavelength peak emission) dye imaged first, using excitation wavelengths that are only minimally absorbed by the skewed spectral tails of the bluer dyes.

• This also minimizes photobleaching from higher-intensity argon laser.

2. Utilize sequential scan instrument settings.• More efficient than manually turning lasers on and off.

Setting up Fluoview for Sequential Scanning to minimize crossover (Manual, 2-71, 72)

1. Select the [Surface XY-Norm] option button in the [Mode] group box in the [Acquire] panel and select <Sequen> from the list displayed below it.

2. Select the observation mode with the option buttons in the [Acquire] panel.

3. [Group] (which means each laser) and the <▲> and <▼> buttons appear on the lower part of each [CH] group box.

4. Set the group number according to the reagent in use with the <▲> and <▼> buttons.

• Set the longer wavelength first to avoid bleaching.

5. Click the <Seq. Once> button to acquire the image.

Controls for Double Labeling

• Background control: specimen without secondary antibody or fluorochrome

• Bleed-through control: specimen labeled with each fluorochrome separately. To determine maximum gain before bleed-through:1. Image green-labeled sample w/488 in Ch. 1, look for

cross-over in Ch. 2.2. Image red-labeled sample w/543 in Ch. 2, look for

crossover in Ch. 1.3. Using these settings, image double-labeled sample

(same stain concentrations as above).

Quantum Dots• Semi-conductor nanocrystals

coated with inert polymer to which biomolecules can be attached, e.g. antibody.

• Advantages:– Less photobleaching, high

quantum yield– Narrow, symmetrical emission

spectra means less spectral overlap.

– Various colors can be excited by same laser line

– Electron-dense, good for correlative LM-EM

• Diadvantages– Toxic (Cadmium Selenide), so

must be used with fixed cells.

Reflection (or Reflectance) Imaging

• Used to image surface topography of sample, especially in materials science.

• Can also be used in labeled or unlabeled biological samples.– Brain, skin, bone, teeth, eye– Probes that can be used in reflected light mode for

single label experiments include gold particles, peroxidase labels, and silver grains.

– Metal-coated SEM samples

• These are confocal images, unlike laser transmitted images.

Illustrated in Figure 3 are several digital images collected from confocal reflection microscopy experiments with silver grains. The specimen is peripheral blood cells from an HIV-infected individual prepared for in-situ hybridization and stained with Giemsa. Figure 3(a) illustrates the preparation under standard brightfield illumination, while Figure 3(b) shows the same field in darkfield illumination with a significant amount of out-of-focus debris. The results with confocal reflection microscopy (of the same viewfield) are presented in Figure 3(c). Note that the out-of-focus debris is not imaged in this mode.

Reflected mode can also provide context to fluorescence image, like laser transmitted DIC

Colloidal Silica Crystal Seeded on Ion Beam Milled Glass SubstrateLeica SP-2 confocal microscope reflection mode image of monodisperse rhodamine core silica microspheres(1um)spontaneously arranged from suspension on a pattern which has been ion-beam milled into the surface of a glass coverslip. This arrangement is designed to form the first layer of a crystalline three dimensional structure. Image Courtesy: Michael Bevan, Ph.D., Beckman Fellow, Beckman Institute for Advanced Science and Technology, UIUC, 61801 http://www.itg.uiuc.edu/exhibits/iotw/2002-02-14/

Diatoms imaged in reflection mode

• From the Laboratory of Research on the Structure of Matter (LRSM), University of Pennsylvania, http://glinda.lrsm.upenn.edu/resolution.html

• Note that the resolution approaches the theoretical limit

TBA, Part 1: Reflection Imaging

• Use a coin supported by a slide, e.g. Lincoln seated in his memorial on a penny.

• Focus with 10X with room lights on.• Set up the confocal for imaging with the HeNe laser and

collecting all light in channel 2. No filters should be used in this case.

• You will need to turn the PMT and Gain way down.• Collect a Z series, using an appropriate step size, and

reconstruct a 3D image.• Save extended focus (lastnameFig1A) and 3D

(lastnameFig1B) images onto 2nd Imaging Computer and submit Figure legend with tech info next week.

TBA, Part 2: Sequential scanning

• Go back to the BPAE cells using the 40X lens. Find a nicely-stained cell and zoom up to 3-4X.

• Optimize your levels as best you can, without changing the laser power, in normal simultaneous scan mode.– Collect a green/red image, save as lastnameFig2A.

• Referring to 2-71 ff. from the manual, set up sequential scanning.– Collect a green/red image, save as lastnameFig2B.

You should note less yellow, indicating less crossover. Include Figure 2 legend with tech info.