Table 1 Optical Parameters for Resolving 3-Color Labeled Cells in an Embryo (A Comparison of Channel vs Lambda Scanning and 2P Microscopy)
From: Multispectral fingerprinting for improved in vivo cell dynamics analysis
Combination | Scan Type | Other Information | |||||||
|---|---|---|---|---|---|---|---|---|---|
3-Color | Channel Mode (3 channel) | Lambda Mode (Spectral Unmixing) | + = good ++ + = best | Single Photon (nm) Excitation Profiles | 2P | 2P Comments | 1P Comments | Â | Â |
Gap-43 GFP & Gap-43 YFP & H2B-mCherry | + separation | ++ separation†| 3 track 488/514/561 2 track 488 + 561/514 1P Lambda 488/561 | Yes | Overlap of GFP/YFP spectra in range 890-990 nm. Difficult to avoid dual excitation. | Reducing tracks to 2 saves time. May use 488 nm GFP/YFP or multiple laser combinations. |  |  | |
Gap-43 CFP & Gap-43 YFP & H2B-mCherry | +++ separation‡ | +++ separation†| 3 track 405 or 458/514/561 2 track 405 or 458/561 + 514 1P Lambda 3 laser | Yes | Excitations to note: 800 nm : minimum YFP ex. 865 nm: max. CFP ex. 950 nm : max. YFP ex. 900 nm: max. dual ex. | May require 3 lasers. Available 1P excitation wavelengths may not be ideal (440 nm) for CFP. |  |  | |
General Data Properties | Example Stack Comparison | ||||||||
Property | Channel* | Lambda | Linear Unmixed | Frame: 1024 × 1024 × 15 μm (42 slice) Interval: 0.36 μm Avg: 4, Speed 7 (dwell 1.6 μs) | |||||
Dataset Sizes (GB) | 0.1 to 1 | 1 to 7 | 1/8th stack size | Â | Size | Scan Time | Save Time | Unmix Time | Unmix Size |
Time to Image (Min./GB) | 10* (1-10 min.) | 8 (8-56 min.) | 5 t unmix Proportional to dataset size | Channel 8-bit 2 track 45s/slice | 160 MB | 22 min | << 1 min | Not Applicable | Not Applicable |
Time to Save (min.) | ≤ 5 | ≈ t acquisition (8-56 min.) | << 1 | Lambda 12-bit 15s/slice | 880 MB | 11 min | 11 min | 4:30 min | 110 MB |
Time to Open (Min./GB) | << 1 | 1.5 | << 1 | Â | Â | Â | Â | Â | Â |
Linear unmixing requires extra processing time. | Linear unmixing time advantages diminished at higher data set sizes | ||||||||
* Channel data for three-track scanning. ‡ Rating based largely on time considerations. †Ratings roughly equivalent, however, CFP/YFP is more widely separated than GFP/YFP, and should generally perform better in unmixing. | |||||||||
Computer properties | Windows Vista. Intel Dual Core Xeon x5260 3.33 MHz, 32-bit. 3 GB RAM. Data (D:) drive capacity 500 GB. Graphics ATI FireGL V5600 512 MB memory. | ||||||||
Objective | LD C-Apochromat 40×/1.1 W Korr | ||||||||
Lambda scan: | Depth (μm) 20 - 45 μm. Frame 1024 × 1024. 12-bit. Frame Avg. 4. Speed 7. Lasers 3% 488, 2.4% 561. Parameter ranges: Frame size: 512-1024, 8-16 bit. Frame avg: 2-8. Scan speed: 7-9. | ||||||||