Table 1.
Summary of imaging parameters (Zeiss LSM510/META/NLO specifications)
| Method |
Laser |
Emission | Imaging parameters | Image |
|---|---|---|---|---|
| Excitation | ||||
| Live Tissue |
860 nm |
390-465 nm |
Ch2 BP 390–465 IR |
Figure 2 |
| (5.8%) |
500-550 nm |
Ch3 BP 500–550 IR |
||
| MBS: HFT KP650 | ||||
| DBS1: Mirror | ||||
| DBS2: NFT 490 | ||||
| FW1: None | ||||
| Live Tissue |
860 nm |
390-465 nm |
ChS BP 393-436 |
Figure 3A-C |
| (5.8%) |
|
ChD |
||
| MBS: HFT KP650 | ||||
| DBS1: Mirror | ||||
| DBS2: NFT 490 | ||||
| FW1: None | ||||
| |
860 nm |
390-465 nm |
ChS BP 393-436 |
Figure 3D |
| (5.8%) |
500-550 nm |
Ch2 BP 500-550 |
||
| ChD | ||||
| MBS: HFT KP650 | ||||
| DBS1: Mirror | ||||
| DBS2: NFT 490 | ||||
| FW1: None | ||||
| Live Tissue |
860 nm |
390-465 nm |
Ch2 BP 390–465 IR |
Figure 4 (Live Tissue) |
| (5.8%) |
500-550 nm |
Ch3 BP 500–550 IR |
||
| ChD |
ChD (unfiltered) |
|||
| MBS: HFT KP650 | ||||
| DBS1: Mirror | ||||
| DBS2: NFT 490 | ||||
| FW1: None | ||||
| Carmine Alum WM |
860 nm |
390-465 nm |
Ch2 BP 390–465 IR |
Figure 4 (Whole Mount) |
| (5.8%) |
500-550 nm |
Ch3 BP 500–550 IR |
||
| ChD |
ChD (unfiltered) |
|||
| MBS: HFT KP650 | ||||
| DBS1: Mirror | ||||
| DBS2: NFT 490 | ||||
| FW1: None | ||||
| Carmine Alum WM |
543 nm (50.5%) |
565-615 nm |
Ch3 BP 565–615 IR |
Figure 5A,B |
| ChD | ||||
| MBS: HFT 488/543/633 | ||||
| DBS1: NFT 635 VIS | ||||
| DBS2: NFT 545 | ||||
| FW1: None | ||||
| Carmine Alum WM |
750 nm (2.1%) |
565-615 nm |
Ch3 BP 565–615 IR |
Figure 5C |
| MBS: HFT KP650 | ||||
| DBS1: NFT KP545 | ||||
| DBS2: NFT 545 | ||||
| FW1: None | ||||
| BF NDD | ||||
| Carmine Alum WM |
750 nm (5.8%) |
565-615 nm |
Ch3: BP 565–615 IR |
Figure 6 |
| MBS: HFT KP650 | ||||
| DBS1: Mirror | ||||
| DBS2: NFT 545 | ||||
| FW1: None | ||||
| Carmine Alum WM |
860 nm (5.8%) |
390-465 nm |
Ch2 BP 390–465 IR |
Figure 7 |
| 650-710 nm |
Ch3 BP 650–710 IR |
|||
| ChD |
ChD (unfiltered) |
|||
| MBS: HFT KP650 | ||||
| DBS1: Mirror | ||||
| DBS2: NFT 490 | ||||
| FW1: None | ||||
| Carmine Alum WM |
860 nm (5.8%) |
393-436 |
ChS BP 393-436 |
Figure 7B |
| ChD |
ChD (unfiltered) |
SHG-B/SHG-F image only |
||
| MBS: HFT KP650 | ||||
| DBS1: Mirror | ||||
| DBS2: NFT 490 | ||||
| FW1: None | ||||
| Carmine Alum WM |
860 nm (5.8%) |
390-465 nm |
Ch2 BP 390–465 IR |
Figure 8 |
| 650-710 nm |
Ch3 BP 650–710 IR |
|||
| ChD |
ChD (unfiltered) |
|||
| MBS: HFT KP650 | ||||
| DBS1: Mirror | ||||
| DBS2: NFT 490 | ||||
| FW1: None | ||||
| Carmine Alum WM |
860 nm (5.8%) |
390-465 nm |
Ch2 BP 390–465 IR |
Figure 9 |
| 650-710 nm |
Ch3 BP 650–710 IR |
|||
| ChD |
ChD (unfiltered) |
|||
| MBS: HFT KP650 | ||||
| DBS1: Mirror | ||||
| DBS2: NFT 490 | ||||
| FW1: None | ||||
| Carmine Alum WM |
750, 800, 830, 860, 890, 950 nm (0.1%) |
Lambda scan |
ChS:361-704 |
Figure 10 |
| 361-704 nm |
MBS: HFT KP650 |
|||
| DBS1: None | ||||
| DBS2: None | ||||
| FW1: None | ||||
| Carmine Alum WM |
860 nm (5.8%) |
390-465 nm |
Ch2 BP 390–465 IR |
Figure 11 (Ex 860/ Em 650–710) |
| 650-710 nm |
Ch3 BP 650–710 IR |
|||
| ChD |
ChD (unfiltered) |
|||
| MBS: HFT KP650 | ||||
| DBS1: Mirror | ||||
| DBS2: NFT 490 | ||||
| FW1: None | ||||
| |
750 nm (5.8%) |
565-615 nm |
Ch3: BP 565–615 IR |
Figure 11 (Ex 750/ Em 565–615) |
| MBS: HFT KP650 | ||||
| DBS1: Mirror | ||||
| DBS2: NFT 545 | ||||
| FW1: None | ||||
| Unstained WM |
735, 860, 960 nm (0.1%) |
Lambda scan |
ChS:361-704 |
Figure 12A, B |
| 361-704 nm |
MBS: HFT KP650 |
|||
| DBS1: None | ||||
| DBS2: None | ||||
| FW1: None | ||||
| |
860 nm (5.8%) |
390-465 nm |
Ch2 BP 390–465 IR |
Figure 12C-E |
| 500-530 nm |
Ch3 BP 500–530 IR |
|||
| or |
or |
|||
| 650-710 nm ChD |
Ch3 BP 650–710 IR ChD (unfiltered) |
|||
| MBS: HFT KP650 | ||||
| |
DBS1: Mirror |
|||
| DBS2: NFT 490 | ||||
| FW1: None | ||||
| Unstained WM |
860, 890 nm (5.8%) |
390-465 nm |
Ch2 BP 390–465 IR |
Additional file 13: Figure S11 |
| 500-530 nm |
Ch3 BP 500–530 IR |
|||
| ChD |
ChD (unfiltered) |
|||
| MBS: HFT KP650 | ||||
| DBS1: Mirror | ||||
| DBS2: NFT 490 | ||||
| FW1: None | ||||
| |
800, 890 nm (0.1%) |
Lambda scan |
ChS:361-704 |
Additional file 13: Figure S11 |
| 361-704 nm |
MBS: HFT KP650 |
|||
| DBS1: None | ||||
| DBS2: None | ||||
| FW1: None | ||||
| Unstained WM |
800, 890 nm (0.1%) |
390-465 nm |
Ch2 BP 390–465 IR |
Additional file 13: Figure S11 |
| 500-530 nm |
Ch3 BP 500–530 IR |
|||
| ChD |
ChD (unfiltered) |
|||
| MBS: HFT KP650 | ||||
| DBS1: Mirror | ||||
| DBS2: NFT 490 | ||||
| FW1: None | ||||
| Carmine Alum WM |
860 nm (5.8%) |
390-465 nm |
Ch2 BP 390–465 IR |
Additional file 1: Figure S1 |
| 650-710 nm |
Ch3 BP 650–710 IR |
|||
| ChD |
ChD (unfiltered) |
|||
| MBS: HFT KP650 | ||||
| DBS1: Mirror | ||||
| DBS2: NFT 490 | ||||
| FW1: None | ||||
| Live Tissue |
860 nm (0.1%) |
Lambda scan |
ChS:361-704 |
Additional file 2: Figure S2 |
| 361-704 nm |
MBS: HFT KP650 |
|||
| DBS1: None | ||||
| DBS2: None | ||||
| FW1: None | ||||
| Carmine Alum WM |
860 nm (5.8%) |
390-465 nm |
Ch2 BP 390–465 IR |
Additional file 4: Figure S3 |
| 650-710 nm |
Ch3 BP 650–710 IR |
|||
| ChD |
ChD (unfiltered) |
|||
| MBS: HFT KP650 | ||||
| DBS1: Mirror | ||||
| DBS2: NFT 490 | ||||
| FW1: None | ||||
| Carmine Alum WM |
860, nm (5.8%) |
390-465 nm |
Ch2 BP 390–465 IR |
Additional file 5: Figure S4 |
| 650-710 nm |
Ch3 BP 650–710 IR |
|||
| ChD |
ChD (unfiltered) |
|||
| MBS: HFT KP650 | ||||
| DBS1: Mirror | ||||
| DBS2: NFT 490 | ||||
| FW1: None | ||||
| Carmine Alum WM |
860 nm |
Lambda scan |
ChS:361-704 |
Additional file 7: Figure S5A,B |
| (0.1%) |
361-704 nm |
MBS: HFT KP650 |
Additional file 11: Figure S9B |
|
| DBS1: None | ||||
| DBS2: None | ||||
| FW1: None | ||||
| Carmine Alum WM |
860 nm (5.8%) |
390-465 nm |
Ch2 BP 390–465 IR |
Additional file 7: Figure S5C |
| 650-710 nm |
Ch3 BP 650–710 IR |
|||
| or |
or |
|||
| 565-615 |
Ch3 BP 565–615 IR |
|||
| ChD |
ChD (unfiltered) |
|||
| MBS: HFT KP650 | ||||
| DBS1: Mirror | ||||
| DBS2: NFT 490 | ||||
| FW1: None | ||||
| Carmine Alum WM |
860 nm (5.8%) |
390-465 nm |
Ch2 BP 390–465 IR |
Additional file 8: Figure S6 |
| 650-710 nm |
Ch3 BP 650–710 IR |
|||
| or |
or |
|||
| 565-615 |
Ch3 BP 565–615 IR |
|||
| ChD |
ChD (unfiltered) |
|||
| MBS: HFT KP650 | ||||
| DBS1: Mirror | ||||
| DBS2: NFT 490 | ||||
| FW1: None | ||||
| Carmine Alum WM |
860 nm (5.8%) |
390-465 nm |
Ch2 BP 390–465 IR |
Additional file 9: Figure S7 |
| 650-710 nm |
Ch3 BP 650–710 IR |
|||
| or |
or |
|||
| 565-615 |
Ch3 BP 565–615 IR |
|||
| ChD |
ChD (unfiltered) |
|||
| MBS: HFT KP650 | ||||
| DBS1: Mirror | ||||
| DBS2: NFT 490 | ||||
| FW1: None | ||||
| Unstained WM |
860 nm |
Lambda scan |
ChS:361-704 |
Additional file 10: Figure S8 |
| (0.1%) |
361-704 nm |
MBS: HFT KP650 |
||
| DBS1: None | ||||
| DBS2: None | ||||
| FW1: None | ||||
| Unstained WM |
860 nm (0.1%) |
390-465 nm |
Ch2 BP 390–465 IR |
Additional file 11: Figure S9A, C |
| 500-530 nm |
Ch3 BP 500–530 IR |
|||
| ChD |
ChD (unfiltered) |
|||
| MBS: HFT KP650 | ||||
| DBS1: Mirror | ||||
| DBS2: NFT 490 | ||||
| FW1: None | ||||
| Unstained WM | 860 nm (0.1%) | 390-465 nm |
Ch2 BP 390–465 IR |
Additional file 12: Figure S10 |
| 500-530 nm |
Ch3 BP 500–530 IR |
|||
| ChD | ChD (unfiltered) |
|||
| MBS: HFT KP650 | ||||
| DBS1: Mirror | ||||
| DBS2: NFT 490 | ||||
| FW1: None |
Filters and Dichroics (LSM 510 Manual).
KP= Low Pass Filter. Example: KP 685 passes wavelengths less than 685 nm to the detector.
LP= High Pass Filter. Example: LP 505 passes wavelengths higher than 505 nm to the detector.
BP= Bandpass Filter. Example: BP 565–615 passes wavelengths 565 nm to 615 nm to the detector.
HFT= Main Dichroic Beam Splitter. Example: HFT 488/543 Excitation wavelengths (488 and 543 nm) sent to the sample.
Passes all other wavelengths up to the detection channels. Example: HFT KP 700/488 Works as both a dichroic and a low pass filter. Will pass all wavelengths less than 700 nm, except for 488 nm, which hits sample, but is not sent to detector.
NFT= Secondary Dichroic Beam Splitter. Example: NFT 543 Wavelengths above 543 nm, pass straight through. Wavelengths less than this amount are reflected 90 degrees.
NT= Neutral Density Filter. Example: Works as an attenuation filter. NT 80/20 passes 80% of the light, cuts 20% of light.