Figure 1.
All the sub-assemblies are indicated including: detector sub-assembly, scanning sub-assembly, condenser sub-assembly, kinematic sub-assembly, xy motor stage sub-assembly, sample stage sub-assembly and table optics sub-assembly. Red line indicates the path of the Ti:Sapph laser. Angled perspective view (top), front view (left) and side view (right) is shown (see Model S1– Microscope Assembly).
Figure 2.
Left) Green circle localizes the detector sub-assembly on TIMAHC. Top) Main parts indicated including: GaAsP PMTs, fluorescence cubes, objective lens and z motorized slider. Bottom) Cross section showing the internal optics: primary and secondary dichroics, green and red emission filters, primary collection lens and PMT aspheric lenses (see Model S1– detector sub-assembly).
Figure 3.
Left) Green circle localizes the scanning sub-assembly on TIMAHC. Right) Main parts of the scanning sub-assembly indicated including: galvanometric scanning mirrors, scan lens, tube lens, sliding cube, lens mag changer and NIR camera. Bottom) Different positions of the sliding mirror cube are shown to select for either two-photon imaging or a LED/camera transmitted image of the sample (see Model S1– scanning sub-assembly).
Figure 4.
Left) Green circle localizes the condenser sub-assembly on TIMAHC. Right) Main parts of the condenser are shown including: condenser lens, field lens, condenser iris, field iris, fine z translator, 50/50 splitter cube, collimation lens, 940 nm LED and NIR sensitive PMT (see Model S1– condenser sub-assembly).
Figure 5.
Left) Green circle localizes the table optics sub-assembly in relation to TIMAHC. Top) Main components of the sub-assembly are shown including: 50/50 beam splitter, imaging shutter, beam dumps, kinematic turning mirror, Pockel’s cell and stand, fixed turning mirror(s) as well as beam expansion and collimation parts (two achromatic doublet lenses and a translator). Red line indicates the path of the Ti:Sapph laser through the table optics (see Model S1– table optics sub-assembly).
Figure 6.
The kinematic mirrors and xy motor stage sub-assemblies.
Left) Green circles localize the kinematic mirrors and xy motor stage sub-assemblies on TIMAHC. Right) Upper and lower kinematic mirrors are shown, along with a turning mirror that directs the beam onto the scanning mirrors. Bottom) xy motor stage and single-axis turning mirror are shown. This allows TIMAHC to move in the xy direction while maintaining beam alignment (see Model S1– kinematic sub-assembly).
Figure 7.
Left) Green circle localizes the sample stage sub-assembly in relation to TIMAHC. Right) Main parts of the stage sub-assembly are shown including: stage top, stage bottom, tissue bath, height adjustable legs and manipulator towers. Bottom) Close up of tissue bath is shown, highlighting sample compartment, input/output channels, over flow trough and over flow drainage hole (see Model S1– stage sub-assembly).
Figure 8.
Microscope Performance: Field, resolution and signal-to-noise.
A) The field of view using the Zeiss 40X NA1.0 objective at a scan angle of 15 degrees was determined by imaging a USAF standard resolution target. The dark bar is from group 1 element 1 on the target, which is 250 microns wide. The field was calculated to be 292 microns wide. B) Point-spread functions showing the axial (left) and radial (right) resolution limits determined by imaging 100 nm Fluosphere beads at 770 nm excitation. C) Fluorescence collection determined either by the PMT generated grey values on a 12 bit image at 60% detector gain (left) or measuring the power of the collected light with a photodiode power meter (right) when exciting the red dye SR-101 at different excitation powers. D) The mean red fluorescence signal and associated SD (left) and the signal to noise ratio: mean/SD (right) of SR-101 imaged at different powers of excitation. Data compares TIMAHC to three commercial two-photon systems: Olympus FV300, Nikon A1 and Leica SP5 II.
Figure 9.
Microscope Performance: Tests for chromatic aberration and radial distortion.
A) Convallaria sample excited at 850 nm collecting green and red fluorescence (small images left) (channels merged right). Lines represent profiles analyzed. B) Green and red structure width (FWHM) and location of peak (Gaussian fit), from line indicated with star in A. C) Summary showing the pixel distance of the green and red FWHM and the pixel distance difference between the two curve fit peaks. D) Convallaria sample excited at 850 nm and 950 nm collecting green fluorescence (small images left) (images merged right). Lines represent profiles analyzed. E) 850 and 950 structure width (FWHM) and location of peak (Gaussian fit), from line indicated with star in D. F) Summary showing the pixel distance of the 850 and 950 FWHM and the pixel distance difference between the two curve fit peaks. G) Sequential images at the full field of view (cropped rectangular), each taken after the stage was moved 45 microns in one linear direction by the motorization. Red arrows point to a particular feature to show the translation across images. H) Expected (dotted line) and measured (symbol) distances of a fine feature as it was moved at 45 micron increments across the full field of view. Segmental (blue circle) and cumulative (purple diamond) measurements were made.
Figure 10.
Microscope Performance: Ca2+ imaging in acutely isolated tissue.
A) Two-photon fluorescence images of brain slices taken from GLAST-Cre LSL-GCaMP3 mice. The expression of the Ca2+ indicator GCaMP3 in astrocytes is shown by colocalization to SR-101. Astrocyte arbor outlined in yellow. B) Astrocyte close up showing micro-domain regions of interest. C) Raw Ca2+ signals from the regions of interest in B. Insert shows the ΔF/F. D) Neurons and astrocytes loaded with Rhod-2/AM (grey) and an arteriole filled with FITC-dextran (green) in the neocortex. Regions of interest shown. E) Raw Ca2+ signals detected from the regions of interest in D, in response to 1s 50 Hz electrical stimulation of afferent fibers. Insert shows the ΔF/F.
Figure 11.
Microscope Performance: In vivo depth vs power and wide-to-small field Ca2+ imaging.
A) Image depth versus power demonstration. Microvasculature filled with FITC-dextran. Four 50 µm thick max intensity stack images taken at different depths in mouse neocortex using a Nikon 16X 0.8NA objective lens, from the surface of the brain down to 950 µm. B) Complete 3D volume stack with a maximum depth of 960 µm (with background subtraction). Left side shows the sections of the stack that are shown in A. Right side shows the imaging depth versus the average excitation power at 780 nm. C) Wide field Ca2+ imaging with the Nikon 16X NA0.8 lens. Astrocytes were bulk loaded with Rhod-2/AM. D) Close field Ca2+ imaging. Single penetrating arteriole, capillaries (green), astrocyte endfoot and processes (grey) using the Zeiss 40X NA1.0 objective lens is shown.
Figure 12.
Microscope Performance: visually guided patch clamp.
A) NIR LED and camera transmitted image of acutely isolated brain slices of the neocortex (left) and the hippocampus (right). B) Transmitted image of the tissue generated by the Ti:Sapph beam captured on the under-stage PMT. Patch pipette and cells can be visualized (left). A successful patch is shown with Alexa 488 fill (right). C) Whole-cell patch clamp of cortical pyramidal neuron (left) and astrocyte (right), each dialyzed with a standard K-Gluconate internal solution containing 100 µM Alexa-488 (Green). Images are displayed as a max projection to capture the cell(s) and the patch pipette.
Figure 13.
Microscope Performance: Under-stage transmitted channel.
A) Transmitted image (left) and green fluorescence (right) of Convallaria standard sample. B) Traces showing fast correlated noise between the green and transmitted channel. The ratio of the two signals eliminates the synchronous noise and reduces the SD in the signal. C) Summary bar graph. Inset shows correlation between the green and transmitted signal r = 0.89. D) Traces showing slow correlated oscillations between the red (SR-101) and transmitted channel in isolated tissue, acquired from region shown in E. The ratio of SR-101/Trans largely removes the undulation and reduces the SD on 5 s binned data, summarized in F. Inset shows correlation between the red and transmitted signal on 5 s binned data (r = 0.97). G) Transmitted images of a small arteriole before (left) and after vasoconstriction (right) caused by the thromboxane receptor agonist U46619 (200 nM). Intrinsic Optical Signals (IOSs) captured by the under-stage PMT in response to synaptic activity (H) and a wave of spreading depression (SD) (I).