PMC

Comparison of fAP amplitudes recorded from granule cell somata at 25°C (n = 12) and 35°C (n = 14). Right: Comparison of f-EPSP amplitudes recorded from somata of granule cells at 25°C (n = 8) and 35°C (n = 10). B. Left: Comparison of fAP width at 25°C and 35°C. Right: Comparison of SNR at 25°C and 35°C. C. Left: Pair-matched comparisons of optically recorded fAP amplitude between the soma and dendritic claws of granule cells (n = 7) recorded at 35°C. Right: Pair-matched comparisons of optically recorded f-EPSP amplitude between the soma and dendritic claws of granule cells (n = 7). D. Left: Pair-matched comparisons of fAP widths between the soma and dendritic claws. Right: Pair-matched comparisons of SNR of f-EPSPs between the soma and dendritic claws.

2P images of HEK-293 cells labeled extracellularly with DiO at excitation wavelengths of 880, 920 and 960 nm. B. Plot of DiO fluorescence intensity across 2P excitation wavelengths in HEK cells (n = 5), normalized to fluorescence obtained at 940 nm. Colored lines and points represent the plots of the individual cells; the black line is the average across cells. C. 2P images of a L5 cortical pyramidal cell patch-labeled with DiO at excitation wavelengths of 880, 920 and 960 nm. D. Plot of DiO fluorescence intensity across 2P excitation wavelengths in two L5 pyramidal cells, normalized to fluorescence obtained at 940 nm. Laser power, measured at the back of the objective, was held constant across all wavelengths.

. Compressed Z-stack of negative images of 4 pyramidal cells (indicated as cells 1,2,3, and 5) and 1 interneuron (cell 4) labeled with DiO via iontophoresis. A whole-cell recording was subsequently made from pyramidal cell 3. The red dashed line outlines the whole-cell pipette. B. Single optical section detailing the optical recording site (arrow) and whole-cell pipette location (red dotted line) on pyramidal cell 3. The cell was patched 40 minutes after loading. C. Average of 40 trials showing the electrical (upper trace) and optical (lower trace) voltage measurements from the cell in B.

Sample confocal images with 1 µm-wide fluorescence line profiles taken from a Purkinje cell (A) and a hippocampal CA1 pyramidal cell (B). Regions of edge fluorescence are indicated by arrows and sample regions of internal fluorescence are indicated by shaded boxes. C-D. Linear fits (solid line) of fAP amplitude vs. ”relative edge fluorescence” with 95% confidence intervals denoted by dashed lines. Circles represent individual data points, with blue points representing the lowest fAP amplitudes and the red points representing the highest fAP amplitudes. Significant correlations are observed in both Purkinje cells (C) and Pyramidal cells (D).

Confocal images of DiO labeling in a Purkinje cell acquired upon sealing with the DiO-filled pipette (left), upon rupturing the membrane (center) and 15 minutes after attaining whole-cell configuration (right). B. Confocal image of a Purkinje cell patch-loaded with DiO. Blue crosses indicate spots where optical measurements were taken in panels D and E. C. Confocal image of the same Purkinje cell with a cross indicating the spot where an axonal measurement was taken. D. Single APs (left) and complex-like spikes (right) recorded either electrophysiologically at the soma (top trace) or optically from different regions within the same cell. As indicated, some optical traces in this and subsequent figures are averaged with number of traces indicated in parentheses. Displayed here are AP and complex spike-like signals, respectively, in the soma (15, 9), in the proximal dendrite (12,12), and in the distal dendrite (8,9). E. The upper trace is the soma electrical recording of membrane voltage, and the trace below is an average of 6 fAPs recorded in the axon.

Single-photon fluorescence emission spectra of DiO (left, green line) and DiB (right, blue line) overlaid with the excitation spectrum of DPA. Overlap between the emission spectra of the fluorescence donors and the FRET acceptor (DPA) is depicted by the shaded areas of each plot. Note the greater overlap between the emission spectrum of DiB and the excitation spectrum of DPA. B. Upper: Confocal image without pinhole of a Purkinje cell patch-labeled with DiB and illuminated with 375 nm laser light. The cross indicates optical recording location. Extracellular fluorescent orbs are out-of-focus dye crystals in the patch pipette. Lower: Optical trace (red) of averaged (12 traces) voltage measurements overlaid with average of simultaneously recorded APs (blue trace) evoked by brief current pulses. C. Comparison of % ΔF/F per AP in cells loaded with DiO (18±2% ΔF/F, 488 nm illumination) and cells loaded with DiB (25±2% ΔF/F, 375–405 nm illumination). D. Comparison of % ΔF/F per 100 mV in cells loaded with DiO (25±3% ΔF/F) and cells loaded with DiB (37±3% ΔF/F). E. Comparison of SNR between cells loaded with DiO (6.7±0.8) and cells loaded with DiB (2.2±0.2). Bars in C-E represent the mean ± SEM.

Negative image of a pyramidal cell membrane labeled with DiO. Dashed lines outline the DiO iontophoretic pipette, withdrawn from the soma after iontophoresis. B. Individual fAPs detected using DiO/DPA in the axon initial segment (arrow in A). The top trace is a somatic whole-cell recording of membrane voltage (Vm), and the middle and bottom traces are successive single trials. Fluorescence traces were not inverted. C. Average of 50 trials showing fAPs and a DiO/DPA optical response to a 6.8 mV depolarization. Data was corrected for photobleaching (<2% decrease ΔF/F over length of trace). D. Vm (top), single trial optical recordings from near the beginning (green) and end (red) of 30 trials, and an average of all 30 trials (bottom trace) from another pyramidal cell. Images were made on soma membrane. No photobleaching correction was necessary in this cell. E. Summary of baseline fluorescence, half width and peak ΔF/F of 1st fAP in the train, and baseline Vm, captured in 3 sets of 10 trails. Colored data points correspond to values measured from individual sweeps in D. Note that values were calculated from unfiltered data.

Confocal image of two GCs patch-labeled with DiO. The blue cross represents the illumination spot on the somatic membrane where the optical measurement was taken. B. Confocal image of a different granule cell patch-labeled with DiO. Blue crosses mark the points where optical measurements of voltage were obtained. C. Expanded view of fluorescence quenching upon depolarization (f-EPSP) by extracellular stimulation of putative single mossy fiber (f-EPSP), overlaid with the stimulus artifact. Note the decreasing slope of the fluorescence change after the end of the stimulus. D. Four single-trial optical recordings (black) of mixed f-EPSPs and fAPs measured from the cell depicted in panel A. The black lines above represent the electrical stimulation (5 pulses at 50 Hz) used to activate single mossy fiber inputs. The red trace below is the average of 5 individual traces where a subthreshold response was followed by 4 fAPs. E. Averaged optical responses (red traces) to a 50 Hz 4-pulse train of stimulation (top, black lines) obtained from the soma (5 traces), dendrite #1 (5 traces), dendrite #2 (4 traces), dendrite #3 (3 traces) and dendrite #4 (7 traces) of the cell displayed in panel B.

Confocal image of CA1 pyramidal cell and detection spot locations where optical measurements were made (crosses). B. Averaged optical traces (in red) recorded from soma (10 traces), primary dendrite (13 traces), secondary dendrite (11 traces) and dendritic spine (11 traces). Blue lines represent electrically recorded APs. In the bottom left is an overlay of three single-trial optical traces recorded from the dendritic spine with a clearly identifiable fAP. C. Confocal image of a different CA1 pyramidal cell basal dendrite and recording spot locations. These experiments were conducted using the confocal pinhole. D. Upper left, blue: Electrical recording of a somatic AP. Below left, red: Averaged, unprocessed fluorescence traces obtained from two points off of the dendrite (point one: 12 traces, and point four: 12 traces), one point on the dendrite (point 2∶17 traces) and one point on the spine (point 3∶24 traces). Note that no significant fluorescence or signal is obtained at points adjacent to the dendritic spine. Right: Optically recorded fAPs normalized to baseline fluorescence recorded in the dendrite (32% ΔF/F max, average of 17 traces) and spine (35% ΔF/F max, average of 24 traces) from the cell pictured in 9C. E. fAP amplitude compared in soma (n = 10) dendrites (n = 8) and spines (n = 5). F SNR compared in soma dendrites and spines. G. fAP width compared in soma dendrites and spines. Bars in E-F represent mean ± SEM.

Sample AP waveforms from three different hippocampal pyramidal cells recorded in control solutions (black), in 2µM DPA (red) and in DPA and DiO together (blue). B. AP amplitude from hippocampal pyramidal cells recorded in control solutions, DPA alone (2 µM), DPA+DiO (2 µM, 1.5 µM, respectively), vehicle control (0.2% DMSO in internal solution), and DiO alone (3 µM). C. AP width for the five experimental groups showing a 77% increase in AP width due to DiO. D. Sample of individual cells’ responses to pulses of -100 (red), +100 (green) and +400 (blue) pA current injection under control conditions, DPA alone, DiO alone and DPA+DiO together. E. Plot displaying number of spikes fired for all 5 groups in response to 200-ms pulses of current injection ranging from -100 to +400pA. F. Comparison of AP amplitude (left) and AP width (right) with and without dye in cerebellar Purkinje cells shows a smaller (34%) broadening of the AP in the presence of DiO. G. Comparison of AP amplitude (left) and AP width (right) with and without DiO in CA1 pyramidal cells recorded at 35°C shows a 35% increase in width and no difference in amplitude due to DiO. H. Comparison of AP amplitude (left) and AP width (right) in cortical pyramidal cells before and after iontophoresis reveal a 53% increase in AP width after dye loading. Bars represent mean ± SEM.