Multi- Photon Microscopy Applications

MULTI-PHOTON APPLICATIONS

• Calcium and Physiology

• Neurobiology

• Developmental Biology

• GFP

• Plants

Calcium and Physiology

Figure 1. Indo-1 Imaging of Ca 2+ changes in motile fish keratocytes This image demonstrates Indo-1 ratio imaging using 2-photon illumination at 700nm. This is a time lapse image showing movement of the keratoctytes and their change of direction when an electric field is applied

Figure 2. Ca 2+ Imaging with NdYLF laser.Time sequence of false colour images showing Ca 2+ changes in developing C. elegans embryo using the non-ratiometric probe, Calcium Green (Molecular Probes)

Figure 1

Acknowledgment:Ingrid Brust-Mascher, Watt Webb, Cornell University. Unpublished image. Permission to use from Watt Webb. Associated Reference: 2-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry. Williams RM, Piston, DW, Webb WW (1994) FASEB Reviews 8, 804-813

Figure 2

Acknowledgment: John White, University of Wisconsin, USA

Unpublished image. Permission to use from J. White.

Neurobiology

Figure 3. Rat hippocampal slice from a 2-days-old rat loaded with the calcium indicator dye fura-2 AM (acetoxymethyl ester). The image shows the CA1 pyramidal cell layer as well as surrounding interneurones. 2-photon excitation allowed fluorometric recordings from individual cell somata and dendrites in the depth of the slice (appr. 40 µm).

Image was taken with an MRC 1024 mounted on an Olympus BX50WI equiped with an 60x (0.9 NA) water-immersed objective. Two-photon excitation was provided by a Ti:Sapphire laser (Spectra Physics) operating at 760 nm, 100 fs, 80 MHz.

Figure 4. Neuron imaging - FITC labelled optic lobe of a moth. A 3D projection of 100 optical sections of FITC-labelled serotonin-immunoreactive neurons in the optic lobe of the moth, Manduca sexta. Imaging was done using 750nm by 2-photon which allowed penetration to a depth of at least 150 mm - deeper than is possible with confocal imaging.

Figure 3

Acknowledgement: Figure 3. Unpublished data from Jens Eilers, Olga Garaschuk, and Arthur Konnerth, I. Physiologisches Institut, Universitaet des Saarlandes

66421 Homburg, Germany

Figure 4.

Acknowledgement: Patricia Jansma, Dept of Neurobiology, University of Arizona with Warren Zipfel, Cornell University. Unpublished image. Permission to use from Watt Webb. A 3D projection of a stack of images taken during the 3D Microscopy course at the University of British Colombia, August, 1996., and imaged at 750 nm using a Zeiss 40x 1.2 NA lens. 100 sections were collected at 1 um steps starting at a depth of ~50 um into the sample. This is deeper into the sample than would be possible by confocal imaging. The cells seen at the lower portion are stained with FITC-antibodies to serotonin. The fibrous structures are part of an autofluorescent tracheole.

Developmental Biology

Figure 5. Multiple label imaging with NdYLF. Triple label image of C. elegans. P Granules (FITC) are shown green, Receptor protein G2P-1 (rhodamine) is shown red. DNA is visualised with DAPI staining by 3-photon excitation.

Developmental Biology

Figures 6 and 7. 2-photon Photoactivation and Lineage tracing in a sea urchin embryo. Left panel shows a 14 hour old L. variegatus embryo where a stripe of cells in the gut have been marked by two-photon photorelease of caged fluorescein (shown in red). No marking has occurred in the outer wall of the embryo, demonstrating the excellent spatial localization of two-photon excitation. Right panel shows the same embryo 34 hours later, when it has become a pluteus larva. The uncaged region has become the oesophagus and front part of the stomach of the larva (shown in red). Pigment cells distributed along the edges of the embryo spontaneously uncage the dye, and are not descendants of the marked region in the gut.

Figure 5.

Acknowledgment: John White, University of Madison Wisconsin, USA Permission. John White and BioImaging Associated Reference:Wokosin, D.L. Centonze, V.E. Crittenden, S. White J.(1996) Three-photon excitation fluorescence imaging of biological specimens using an all-solid-state laser. Bioimaging 4, 208-214

Figure 6 and 7.

Acknowledgement D. Piston, Vanderbilt University R. Summers SUNY, Buffalo. Image is from Piston Webb site. Permission granted. Associated Reference:Summers, RG, Piston DW, Harros KM, Morril JB (1996) The orientation of first cleavage in the sea urchin embryo , Lytechinus variegatus, does not specify the axes of bilateral symmetry. Dev. Biol. 175, 177-183

Green Fluorescence Protein

Figure 8. 2-photon imaging of Green Fluorescent Protein. Three dimensional rendering of a group of tobacco suspension cells with Green Fluorescent Protein (GFP) labeled mitochondria produced from 40 optical sections collected using two photon excitation fluorescence microscopy. Mitochondria were labelled by the use a GFP-mitochondrial transit peptide fusion protein with accumulates GFP in the mitochondria. Excitation from a Spectra-Physics Tsunami Ti:S laser was at 770 nm with a 90fs pulse width. DAPI and GFP fluorescence were collected through a 440 nm bandpass filter (30 nm FWHM) and a 550nm bandpass (60 nm FWHM), respectively.

Figure 8.

Acknowledgment: Warren Zipfel in collaboration with Rainer Kohler and Maureen Hanson, Section of Genetics and Development, Cornell University. Permission. Watt Webb.

Associated Reference:

Kohler RH, Cao J, Zipfel WR, Webb WW, Hanson MR (1997)

Exchange of protein molecules through connections between higher plant plastids Science 276, 2039-2042

PlantsFigure 9. Plant Meiosis - Imaging live cells over a long

period of time. 2-photon imaging can be used to study developing cells over long periods of time without damage. A single optical section taken approximately 150 mm deep in a multiple stained, intact Petunia flower bud. A set of experiments followed the process of meiosis in plants to determine the mechanism of the mitochrondrially-inherited cytoplasmic male sterility (CMS) in plants. Each panel is a merge of two channels of raw data, rhodamine 123 fluorescence (560 - 630nm, green pseudocolor) coming from the mitochondria and DAPI, a nuclear stain which fluoresces at about 460nm (yellow pseudocolor). The excitation was at 780nm and non-descanned epifluoresecence was collected through a 40x 0.9 NA lens. In the left hand side cells undergoing meiosis can be seen (e.g the cell about 1/4 of the way down from the upper left hand corner is in metaphase). The right hand side of the image, marked male sterility shows the same region of the tissue at the same developmental stage, but cell division is never seen since these plants are arrested in prophase and do not produce pollen.

Figure 9.

Permission. Watt Webb. Associated Reference.

Maiti S, Shear JB, Williams RM, Zipfel WR, Webb W W (1997)

Measuring serotonin distribution in live cells with three-photon

excitation. Science 275, 530-532

Autofluorescence

Figures 10 and 11. Serotonin distribution in live cells. Images of rat basophilic leukemia (RBL-2H3) cells obtained from 3-photon fluorescence of serotonin and other cellular components.

Figures 10 and 11.

Acknowledgement: Warren R. Zipfel in collaboration with Catherine Conley and Maureen Hanson, Section of Genetics and Development, Cornell University.

Permission. Watt Webb

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