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a crystal -clear zebrafish for in vivo imaging
by：LCD Mall
2020-06-24
Zebra fish larvae (Danio rerio)
It is an excellent spinal model for in vivo imaging of biological phenomena at the levels of subcells, cells and systems.
However, even in this animal model, the optical accessibility of high-pigment tissues such as eyes is limited.
A typical strategy to improve the transparency of the larvae of the zebrafish fish requires the use of highly toxic compounds (e. g. 1-phenyl-2-thiourea, PTU)
Or a mutant strain of pigmentation (e. g. casper mutant).
So far, none of these strategies have produced larvae that behave normally in both body and eye transparency.
Here we show The Crystal, an optical clear zebrafish Mutation obtained by combining different feasible mutations that affect skin pigmentation.
Compared with the previously described combination mutation casper, the Crystal mutation also lacks pigmentation in the retinal pigment epithelial, thus being able to enter the eye optical. Unlike PTU-
In treated animals, Crystal larvae are able to perform visually guided behaviors as effectively as wild-type larvae, such as optomotor reactions.
In order to verify the in vivo application of Crystal larvae, we conducted a fullbrain light-
Paper imaging and two-
Photon calcium imaging of retinal nerve activity.
In conclusion, this new combination pigmentation mutant represents an ideal spinal tool that can conduct completely unobstructed in-vivo study of the structure and function of biological processes, especially within the eyes or the eyes
The fish is maintained at 28.
In Danieau solution [5 ° c on/10 hours off at 14 hours]58u2009mM NaCl, 0. 7u2009mM KCl, 0. 4u2009mM MgSO, 0. 6u2009mM Ca(NO)5.
0 mM crane skin, pH 7. 6].
Combination mutant (; ; )
Three different single mutant strains were produced by cross-breeding in sequence, namely (ref. ), (ref. )and (ref. )mutants.
It is important that both larvae and adult fish are feasible, and no visible morphological, functional or behavioral abnormalities are shown except for the pigmentation phenotype.
As mentioned earlier, mutations are obtained by hybridization and mutation.
Get wild zebrafish fish with this line.
The genetically modified lines used in this study included and.
A co-focused functional imaging experiment was performed in the mutant. Light-
Thin slice imaging experiments were carried out in and mutant. Two-
Photon functional imaging experiments were performed on larvae.
To treat zebra larvae with PTU, we followed the standard procedure, specifically raising larvae in 200 μm PTU (Sigma)
In Danieau solution within 24 hours after fertilization (hpf). ()
The larvae used for co-focused functional imaging of visual-induced neural activity were treated with 200 μm PTU of 24 hpf to 3 dpf, and were imaged at 4 dpf.
The work was approved by the local animal welfare and ethical review agency (
King\'s College London)
And follow.
According to the permission of the British interior ministry (PPL70/8057). Whole-
Animal images of adult zebrafish fish were taken with a Nikon D7000 digital SLR camera equipped with Sigma macro lenses.
Adult zebrafish fish was given 0 anesthesia. 2% tricaine (MS222, Sigma)
In the fish facility water, put in a 90mm petri dish containing the fish facility water.
The larvae were imaged using Zeiss Axioskop microscope connected to the EXi blue CCD camera (Retiga)
Acquisition of software (PerkinElmer).
Use 0 anesthesia for young fish.
Add 02% of the three aine in Danieau solution and fix it in 1% of the low melting point glue (Sigma)
On the slide
Imaging was performed using a Zeiss m6 710 co-focal microscope equipped with a spectral detection scanner and 20X/1. 0 NA water-
Immersion goals (Carl Zeiss).
Function Time-
Visual-induced calcium response series of retinal cells (RGCs)
Obtained at a speed of 4. 1u2009Hz and 0. 415u2009×u20090.
Resolution of Resolution μ m (
256 pixel x Pixel 256)
, And 1 AU Small hole aperture.
Excitation light is more than 488 nmline laser. Non-
Fix the larvae after anesthesia in a low melting point glue of 2% (Sigma)
Prepared in Danieau solution and installed on the back side placed on a custom glass platformmade Danieau-filled chamber.
Agarose is sufficient to inhibit larvae and therefore does not require anesthesia.
Imaging was carried out in the afternoon (1–8 pm). Whole-brain light-
Thin slice imaging using Zeiss light sheet Z.
One microscope with two 10 x/0.
2 NA lighting targets and a 20X/1. 0 NA water-
Soaking detection target (Carl Zeiss).
The GCaMP6f fluorescence was stimulated by a 488 nm laser excitation light, and the emission light detection was performed using a 505-545 BP filter.
Pivot scanner (Carl Zeiss)
Used to provide even lighting, so avoid shading along the lighting axis.
The film thickness is 5.
Center 39 μm and 10.
8 μm at the edge of the field of view.
Exposure time 29. 97u2009ms.
Big Volume Image 623 u2009 × u2009 798 u2009 × u2009 283 u2009 mu m (
1500 u2009 × u2009 1920 u2009 × u2009 490 pixel)
The resolution is 0. 415u2009×u20090. 415u2009×u20090. 631u2009μm.
4 dpf and larvae were first paralyzed in alpha-for 10-15 minutesbungarotoxin (1u2009mg/ml; Biotium)
Preparation in Danieau solution.
Subsequently, the larvae were fixed in a low melting point glue of 2% (Sigma)
In a glass capillary tube (
Volume 20 μ l, 701904; Brand).
Subsequently, we squeezed out the part of the agar cylinder containing the head of the larvae from the capillary, and targeted the larvae so that the back side of the head was against the detection target, and the eyes faced two lighting targets. Whole-brain light-
The mutant larvae of Zhang image were tested and customized. made light-
Dr. Martin Meyer (
King\'s College London)
With 20X/1. 0 NA water-
Immersion XLUMPlanFLN detection target (Olympus). Two-
Retina surgery for photon functional imaging is equipped with a Nikon A1R million pixel microscope for one month-
Channel GaAsP NDD and Apochromat 25X/1. 1 NA water-
Immersion goals (Nikon).
Chameleon Ultra II mode provides incentives-Locking titaniumSapphire laser (Coherent)
Tuned to 930 nm. Time-
A series of visual-induced calcium reactions were obtained at 4 hz and 0 rates. 248u2009×u20090.
248 μm resolution (
512 pixel x Pixel 256).
After activating the laser scan, we waited for 60 seconds and then started the visual stimulation to ensure that the retina was adapted by multiple-photon laser.
4 dpf larvae were first paralyzed in alpha-for 10-15 minutesbungarotoxin (1u2009mg/ml; Biotium)
Preparation in Danieau solution.
Subsequently, the larvae were fixed in a low melting point glue of 2% (Sigma)
And installed on a raised custom
Glass platform facing up on the back (45° angle tilt)
An eye facing the LCD screen (
See visual stimulation)
Under a custommade Danieau-filled chamber.
Imaging was carried out in the afternoon (1–8 pm).
As mentioned earlier, a stimulus for moving bars is generated.
Diffusion filter (3026, Rosco)
Glued to one side of the room and used as a projection screen.
The Agar in front of the eye facing the projection screen is removed, allowing for unobstructed viewing of the projected image on the side of the chamber.
The larvae are placed 3 cm from the screen and the projected image is full ~ 97 ° × 63 ° view.
Visual stimuli include light (56u2009cd/m)or dark bars (8u2009cd/m)(
175% and 25% of average brightness)
In an average gray background (32u2009cd/m).
Since the quality difference between light and dark strips was not noted, the data obtained using these two stimuli were combined.
The width of each bar is 10 °, moving at a speed of 20 °/s, separated from the previous bar by 30 °, and multiple bars can be displayed on the screen at any time.
The long axis of the rod is orthogonal to the direction of motion.
Each of the 12 movement directions is presented once (3u2009seconds)in a pseudo-
Random Order unique to each slice in each animal imaging. Each inter-
In order for the gcamp5 G signal to return to the baseline, the epoch interval is 10 seconds. A blank-
The empty screen condition for 2 seconds is also staggered.
Experiment with self-defined generation and control vision
Written Labview and MATLAB code (MathWorks)
, Implemented on a visual stimulus presenter (
Cambridge research system
Delivered via DLP Pico projector (Optoma).
Exciting in two motion grating
The photon preparation is generated and controlled using PsychoPy and transmitted via the LCD screen (SKD5VA-
Moon, Hi-Tech)
Under the custom
Made an organic glass room. A long-
Red glass filter (
Thorlabs FGL610)
Located between the LCD screen and the chamber, allowing simultaneous imaging and visual stimulation.
The larvae are placed 2 cm away from the screen and the images on the LCD screen are full ~ 14 ° × 100 ° View (
Average background brightness 30. 4u2009cd/m).
Visual stimuli are made up of squares. wave gratings (
100% contrast, space frequency 1.
66 cycles/cm, 1 cycle/second of time frequency).
8 grating bars per.
The width is 5 °, and the long axis of the rod is orthogonal to the direction of motion.
Each of the 12 movement directions is presented once (6u2009seconds)with and inter-
The 10-second epoch interval that causes the GCaMP6f signal to return to the baseline. A blank-
The screen empty condition for 6 seconds is also staggered. TTL triggers (0-5-0u2009Volts)
Record the era time events generated by the LabJack USB data acquisition device (U3-
LV from LabJack company).
After activating the laser scan, we waited for 60 seconds and then started the visual stimulation to ensure that the retina was adapted by multiple-photon laser.
Five dpf larvae of the individual were located in a 35mm petri dish containing Danieau solution.
LCD screen for IPhone 5 (Apple)
Controlled by MacBook Pro (Apple)
Display by duet (
Kelos technology)
For displaying black and white squares-wave gratings (
85% contrast, space frequency 0.
33 cycle/mm, time frequency 3. 5 cycles/s)
Move in 4 directions (
Angle distance 90 °)
At the bottom of the Petri dish.
Visual stimulation in Keynote (Apple).
A total of 5 tests per larvae (
Each test lasts 6 s, followed by 10 s of the static grating)
And according to the test score of its response (i. e.
, The fish moves and swims in the direction of moving the grating).
Visual monitoring of the behavior of larvae using the M165 FC stereo microscope (Leica).
Calcium imaging data are analyzed as mentioned earlier.
All in all, function Time-
Before the analysis, the series was processed as follows: time-
The series of images for each experiment are rigid-Body algorithm (SPM12; )
, Median filtering with kernel size of 1-body element to remove dark noise and bulk noise, and spatial smoothing with 2D Gaussian, where f = raw original fluorescence)
Change of normal signal strength [%ΔF/Fu2009=u2009(F-B)/B]
Calculated at each body element.
Delta f is used for population function data (voxel-wise analysis)
, While % Δf/F is used for the area of interest defined manually (ROIs).
For each element or ROI, the integral response on the epoch-
Calculate intervals to provide a single response metric for each stimulus motion direction.
The integral in each epoch window is a summary indicator that is more resistant to calcium probe saturation effect than the maximum signal change.
The threshold value of each individual element in the acquisition image sequence is determined according to the variance of the Delta f change between frames
Epoch intervals and empty states, operating systems such as OSI operations> ios0. 5 and DSIu2009 0, respectively. 8;
Therefore, the curve of the fit explains at least 80% of the integral response. A single von-
The Mises distribution is used to fit the response of DS voxels and estimate their preferred motion angle direction from the center of the fitting curve.
The sum of two von-Mises (
180 ° angle distance apart)
Used to fit the response of OS voxels and estimate the preferred direction of its motion angle from the center of the fitting curve.
The cyclic variance is also calculated as an alternative index for orientation selectivity (
Loop variable threshold function in image j and the analyze> histogram> list command.
The obtained value is then multiplied by the volume of the single element (0. 415u2009×u20090. 415u2009×u20090. 631u2009μmu2009=u20091. 086u2009×u200910u2009μm).
Statistical test results are reported in the form of numbers and graphic legends.
Statistical analysis and testing using Prism 6 (GraphPad)
Or MATLAB r204b (MathWorks).
Descriptive statistics before performing statistical tests (e. g.
, Normal test, see if the value is from the Gaussian distribution or F-
Test of comparative variance
Used to select the appropriate statistical test (
Report in figure legend with test results attached).
The standard for statistical significance was set at p
It is an excellent spinal model for in vivo imaging of biological phenomena at the levels of subcells, cells and systems.
However, even in this animal model, the optical accessibility of high-pigment tissues such as eyes is limited.
A typical strategy to improve the transparency of the larvae of the zebrafish fish requires the use of highly toxic compounds (e. g. 1-phenyl-2-thiourea, PTU)
Or a mutant strain of pigmentation (e. g. casper mutant).
So far, none of these strategies have produced larvae that behave normally in both body and eye transparency.
Here we show The Crystal, an optical clear zebrafish Mutation obtained by combining different feasible mutations that affect skin pigmentation.
Compared with the previously described combination mutation casper, the Crystal mutation also lacks pigmentation in the retinal pigment epithelial, thus being able to enter the eye optical. Unlike PTU-
In treated animals, Crystal larvae are able to perform visually guided behaviors as effectively as wild-type larvae, such as optomotor reactions.
In order to verify the in vivo application of Crystal larvae, we conducted a fullbrain light-
Paper imaging and two-
Photon calcium imaging of retinal nerve activity.
In conclusion, this new combination pigmentation mutant represents an ideal spinal tool that can conduct completely unobstructed in-vivo study of the structure and function of biological processes, especially within the eyes or the eyes
The fish is maintained at 28.
In Danieau solution [5 ° c on/10 hours off at 14 hours]58u2009mM NaCl, 0. 7u2009mM KCl, 0. 4u2009mM MgSO, 0. 6u2009mM Ca(NO)5.
0 mM crane skin, pH 7. 6].
Combination mutant (; ; )
Three different single mutant strains were produced by cross-breeding in sequence, namely (ref. ), (ref. )and (ref. )mutants.
It is important that both larvae and adult fish are feasible, and no visible morphological, functional or behavioral abnormalities are shown except for the pigmentation phenotype.
As mentioned earlier, mutations are obtained by hybridization and mutation.
Get wild zebrafish fish with this line.
The genetically modified lines used in this study included and.
A co-focused functional imaging experiment was performed in the mutant. Light-
Thin slice imaging experiments were carried out in and mutant. Two-
Photon functional imaging experiments were performed on larvae.
To treat zebra larvae with PTU, we followed the standard procedure, specifically raising larvae in 200 μm PTU (Sigma)
In Danieau solution within 24 hours after fertilization (hpf). ()
The larvae used for co-focused functional imaging of visual-induced neural activity were treated with 200 μm PTU of 24 hpf to 3 dpf, and were imaged at 4 dpf.
The work was approved by the local animal welfare and ethical review agency (
King\'s College London)
And follow.
According to the permission of the British interior ministry (PPL70/8057). Whole-
Animal images of adult zebrafish fish were taken with a Nikon D7000 digital SLR camera equipped with Sigma macro lenses.
Adult zebrafish fish was given 0 anesthesia. 2% tricaine (MS222, Sigma)
In the fish facility water, put in a 90mm petri dish containing the fish facility water.
The larvae were imaged using Zeiss Axioskop microscope connected to the EXi blue CCD camera (Retiga)
Acquisition of software (PerkinElmer).
Use 0 anesthesia for young fish.
Add 02% of the three aine in Danieau solution and fix it in 1% of the low melting point glue (Sigma)
On the slide
Imaging was performed using a Zeiss m6 710 co-focal microscope equipped with a spectral detection scanner and 20X/1. 0 NA water-
Immersion goals (Carl Zeiss).
Function Time-
Visual-induced calcium response series of retinal cells (RGCs)
Obtained at a speed of 4. 1u2009Hz and 0. 415u2009×u20090.
Resolution of Resolution μ m (
256 pixel x Pixel 256)
, And 1 AU Small hole aperture.
Excitation light is more than 488 nmline laser. Non-
Fix the larvae after anesthesia in a low melting point glue of 2% (Sigma)
Prepared in Danieau solution and installed on the back side placed on a custom glass platformmade Danieau-filled chamber.
Agarose is sufficient to inhibit larvae and therefore does not require anesthesia.
Imaging was carried out in the afternoon (1–8 pm). Whole-brain light-
Thin slice imaging using Zeiss light sheet Z.
One microscope with two 10 x/0.
2 NA lighting targets and a 20X/1. 0 NA water-
Soaking detection target (Carl Zeiss).
The GCaMP6f fluorescence was stimulated by a 488 nm laser excitation light, and the emission light detection was performed using a 505-545 BP filter.
Pivot scanner (Carl Zeiss)
Used to provide even lighting, so avoid shading along the lighting axis.
The film thickness is 5.
Center 39 μm and 10.
8 μm at the edge of the field of view.
Exposure time 29. 97u2009ms.
Big Volume Image 623 u2009 × u2009 798 u2009 × u2009 283 u2009 mu m (
1500 u2009 × u2009 1920 u2009 × u2009 490 pixel)
The resolution is 0. 415u2009×u20090. 415u2009×u20090. 631u2009μm.
4 dpf and larvae were first paralyzed in alpha-for 10-15 minutesbungarotoxin (1u2009mg/ml; Biotium)
Preparation in Danieau solution.
Subsequently, the larvae were fixed in a low melting point glue of 2% (Sigma)
In a glass capillary tube (
Volume 20 μ l, 701904; Brand).
Subsequently, we squeezed out the part of the agar cylinder containing the head of the larvae from the capillary, and targeted the larvae so that the back side of the head was against the detection target, and the eyes faced two lighting targets. Whole-brain light-
The mutant larvae of Zhang image were tested and customized. made light-
Dr. Martin Meyer (
King\'s College London)
With 20X/1. 0 NA water-
Immersion XLUMPlanFLN detection target (Olympus). Two-
Retina surgery for photon functional imaging is equipped with a Nikon A1R million pixel microscope for one month-
Channel GaAsP NDD and Apochromat 25X/1. 1 NA water-
Immersion goals (Nikon).
Chameleon Ultra II mode provides incentives-Locking titaniumSapphire laser (Coherent)
Tuned to 930 nm. Time-
A series of visual-induced calcium reactions were obtained at 4 hz and 0 rates. 248u2009×u20090.
248 μm resolution (
512 pixel x Pixel 256).
After activating the laser scan, we waited for 60 seconds and then started the visual stimulation to ensure that the retina was adapted by multiple-photon laser.
4 dpf larvae were first paralyzed in alpha-for 10-15 minutesbungarotoxin (1u2009mg/ml; Biotium)
Preparation in Danieau solution.
Subsequently, the larvae were fixed in a low melting point glue of 2% (Sigma)
And installed on a raised custom
Glass platform facing up on the back (45° angle tilt)
An eye facing the LCD screen (
See visual stimulation)
Under a custommade Danieau-filled chamber.
Imaging was carried out in the afternoon (1–8 pm).
As mentioned earlier, a stimulus for moving bars is generated.
Diffusion filter (3026, Rosco)
Glued to one side of the room and used as a projection screen.
The Agar in front of the eye facing the projection screen is removed, allowing for unobstructed viewing of the projected image on the side of the chamber.
The larvae are placed 3 cm from the screen and the projected image is full ~ 97 ° × 63 ° view.
Visual stimuli include light (56u2009cd/m)or dark bars (8u2009cd/m)(
175% and 25% of average brightness)
In an average gray background (32u2009cd/m).
Since the quality difference between light and dark strips was not noted, the data obtained using these two stimuli were combined.
The width of each bar is 10 °, moving at a speed of 20 °/s, separated from the previous bar by 30 °, and multiple bars can be displayed on the screen at any time.
The long axis of the rod is orthogonal to the direction of motion.
Each of the 12 movement directions is presented once (3u2009seconds)in a pseudo-
Random Order unique to each slice in each animal imaging. Each inter-
In order for the gcamp5 G signal to return to the baseline, the epoch interval is 10 seconds. A blank-
The empty screen condition for 2 seconds is also staggered.
Experiment with self-defined generation and control vision
Written Labview and MATLAB code (MathWorks)
, Implemented on a visual stimulus presenter (
Cambridge research system
Delivered via DLP Pico projector (Optoma).
Exciting in two motion grating
The photon preparation is generated and controlled using PsychoPy and transmitted via the LCD screen (SKD5VA-
Moon, Hi-Tech)
Under the custom
Made an organic glass room. A long-
Red glass filter (
Thorlabs FGL610)
Located between the LCD screen and the chamber, allowing simultaneous imaging and visual stimulation.
The larvae are placed 2 cm away from the screen and the images on the LCD screen are full ~ 14 ° × 100 ° View (
Average background brightness 30. 4u2009cd/m).
Visual stimuli are made up of squares. wave gratings (
100% contrast, space frequency 1.
66 cycles/cm, 1 cycle/second of time frequency).
8 grating bars per.
The width is 5 °, and the long axis of the rod is orthogonal to the direction of motion.
Each of the 12 movement directions is presented once (6u2009seconds)with and inter-
The 10-second epoch interval that causes the GCaMP6f signal to return to the baseline. A blank-
The screen empty condition for 6 seconds is also staggered. TTL triggers (0-5-0u2009Volts)
Record the era time events generated by the LabJack USB data acquisition device (U3-
LV from LabJack company).
After activating the laser scan, we waited for 60 seconds and then started the visual stimulation to ensure that the retina was adapted by multiple-photon laser.
Five dpf larvae of the individual were located in a 35mm petri dish containing Danieau solution.
LCD screen for IPhone 5 (Apple)
Controlled by MacBook Pro (Apple)
Display by duet (
Kelos technology)
For displaying black and white squares-wave gratings (
85% contrast, space frequency 0.
33 cycle/mm, time frequency 3. 5 cycles/s)
Move in 4 directions (
Angle distance 90 °)
At the bottom of the Petri dish.
Visual stimulation in Keynote (Apple).
A total of 5 tests per larvae (
Each test lasts 6 s, followed by 10 s of the static grating)
And according to the test score of its response (i. e.
, The fish moves and swims in the direction of moving the grating).
Visual monitoring of the behavior of larvae using the M165 FC stereo microscope (Leica).
Calcium imaging data are analyzed as mentioned earlier.
All in all, function Time-
Before the analysis, the series was processed as follows: time-
The series of images for each experiment are rigid-Body algorithm (SPM12; )
, Median filtering with kernel size of 1-body element to remove dark noise and bulk noise, and spatial smoothing with 2D Gaussian, where f = raw original fluorescence)
Change of normal signal strength [%ΔF/Fu2009=u2009(F-B)/B]
Calculated at each body element.
Delta f is used for population function data (voxel-wise analysis)
, While % Δf/F is used for the area of interest defined manually (ROIs).
For each element or ROI, the integral response on the epoch-
Calculate intervals to provide a single response metric for each stimulus motion direction.
The integral in each epoch window is a summary indicator that is more resistant to calcium probe saturation effect than the maximum signal change.
The threshold value of each individual element in the acquisition image sequence is determined according to the variance of the Delta f change between frames
Epoch intervals and empty states, operating systems such as OSI operations> ios0. 5 and DSIu2009 0, respectively. 8;
Therefore, the curve of the fit explains at least 80% of the integral response. A single von-
The Mises distribution is used to fit the response of DS voxels and estimate their preferred motion angle direction from the center of the fitting curve.
The sum of two von-Mises (
180 ° angle distance apart)
Used to fit the response of OS voxels and estimate the preferred direction of its motion angle from the center of the fitting curve.
The cyclic variance is also calculated as an alternative index for orientation selectivity (
Loop variable threshold function in image j and the analyze> histogram> list command.
The obtained value is then multiplied by the volume of the single element (0. 415u2009×u20090. 415u2009×u20090. 631u2009μmu2009=u20091. 086u2009×u200910u2009μm).
Statistical test results are reported in the form of numbers and graphic legends.
Statistical analysis and testing using Prism 6 (GraphPad)
Or MATLAB r204b (MathWorks).
Descriptive statistics before performing statistical tests (e. g.
, Normal test, see if the value is from the Gaussian distribution or F-
Test of comparative variance
Used to select the appropriate statistical test (
Report in figure legend with test results attached).
The standard for statistical significance was set at p
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