In a single-objective light-sheet (SOLS) microscope there are more optics on the emission path compared to a regular microscope. So how does the transmission of a SOLS microscope compare to a standard microscope? Or similar microscopes like OPM, eSPIM or Lattice?
Here's a simple analytical estimate for the transmission of a SOLS (or similar) microscope:
The original SOLS design from 2019 transmits about ~50% of the light of the equivalent widefield epifluorescence microscope (the 'Benchmark'). The 'SOLS re-optimised' configuration with ~65% transmission can be achieved by swapping out the original lens-galvo-lens scanner for a 'lensless' scanner (like a pair of larger galvos), and optimizing the tilt of microscope 3 from 30deg to 25deg. The larger scanning optics in this configuration are typically slower, but should still be faster than sample scanning (e.g. the Lattice).
Microscope | Type | hemispheric transmission | normalized transmission |
---|---|---|---|
Benchmark | Coverslip | 54.23% | 100.00% |
SCAPE, Bouchard 2015 | Dipping | 1.29% | 2.38% |
SOPi, Kumar 2018 | Dipping | 2.46% | 4.54% |
SCAPE, Voleti 2019 | Dipping | 5.54% | 10.22% |
Crossbill, Kumar 2021 | Dipping | 7.04% | 12.99% |
Lattice, Chen 2014 | Dipping | 35.14% | 64.79% |
OPM, Dunsby 2008 | Coverslip | 2.58% | 4.76% |
OPM, Kumar 2011 | Coverslip | 9.76% | 17.99% |
eSPIM, Yang 2019 | Coverslip | 24.75% | 45.63% |
Lattice Zeiss | Coverslip | 27.34% | 50.41% |
SOLS, Millett-Sikking 2019 | Coverslip | 28.87% | 53.23% |
SOLS re-optimised | Coverslip | 36.39% | 67.10% |
The analysis is limited to microscopes that respect a planar boundary for compatibility with standard slides, dishes and multiwell plates (i.e. no additional optics above the coverslip, or below the front face of the objective for dipping systems). In this regime the maximum available light from a spherical emitter is a hemisphere of rays. The estimate for transmission can then be split into:
The total transmission of the system \(T\) is then the product of the ray and optic transmissions: \[ T = T_{rays} \; T_{optics} \tag{1}\]
Ideally behavior is assumed from the objectives. The combined ray transmission is found with: \[ T_{rays} = T_1 \; T_2 \; T_3 \tag{2}\]
If we have a primary objective with a collection half angle \(\theta_1\), then the fraction of rays we can transmit from a hemisphere \(T_1\) is: \[ T_1 = 1 - \cos\theta_1 \tag{3}\] where, \[ \theta_1 = \arcsin{\left ( \frac{NA_1}{n_1} \right )} \tag{4}\] and \(NA_1\) and \(n_1\) are the numerical aperture and refractive index of the immersion of objective 1. Equation (3) is found by taking the ratio of the area of a spherical cap to that of a hemisphere:
We will impose the 'perfect 3D imaging' condition from the original remote refocus paper (Botcherby 2007) and apply the preservation of angles between the object and the remote space (i.e. \(\theta_{1ray} = \theta_{2ray}\)). In this regime if \(\theta_2\) is the collection half angle of objective 2 then we have two possible results for the transmission \(T_2\):
In contrast to a 'straight through' remote refocus, in a SOLS style microscope (Millett-Sikking 2019) objective 3 (with collection half angle \(\theta_3\)) is tilted away from the primary optic axis by an angle \(\theta_t\). The tilting can be thought of as tilting the spherical transmission cap of objective 3 away from the spherical transmission cap of objective 2, which gives rise to four possible conditions:
The combined optic transmission is simply found with: \[ T_{optics} = (T_{ob} \; T_{tl})^{n_{ob}} \; T_{sl}^{n_{sl}} \; T_{mi}^{n_{mi}} \; T_{di}^{n_{di}} \; T_{fi}^{n_{fi}} \tag{11}\] where \(T_{ob}\), \(T_{tl}\), \(T_{sl}\), \(T_{mi}\), \(T_{di}\) and \(T_{fi}\) are the nominal transmission efficiencies for objectives (87.5%), tube lenses (97.5%), scan lenses (90%), mirrors (99%), dichroics (95%) and filters (95%) respectively, and the exponents are the number of each element in the emission path (for example \(n_{mi} = 3\) would be 3 mirrors). The numbers in brackets are the typical transmission values at ~532nm that were used in the provided script (SOLS_transmission.py).
With a 'planar boundary' constraint the ideal microscope would collect hemispheric rays at 100% efficiency. In practice a standard epifluorescence microscope has minimal emission path optics (i.e. maximum transmission) which provides a sensible 'benchmark' for comparison:
Here the Nikon 100x1.35 Silicone oil objective, together with a single tube lens, dichroic and emission filter was used to represent the 'best in class' microscope. The 100x1.35 Sil has one of the highest angular collections currently available and is optimally refractive index matched for in vivo 3D imaging.