Abstract: Methods include generating a modulated light pattern using a light modulator (108), transmitting the modulated light pattern through an objective lens (104) to a surface of a substrate (150), where the objective lens (104) defines an object plane, obtaining a plurality of images of the modulated light pattern reflected from the surface of a substrate (150), where each modulated light pattern is obtained at a different relative distance between the objective lens (104) and the substrate surface, analyzing the plurality of images to determine a reference location of the substrate surface relative to the objective lens (104) that corresponds to positioning of an image of the modulated light pattern formed by the objective lens (104) at the object plane, and adjusting a position of the substrate (150) relative to the objective lens (104) based on the reference location.

Abstract: The disclosure features methods that include exposing a biological sample to illumination light and measuring light emission from the sample to obtain N sample images, where each sample image corresponds to a different combination of a wavelength band of the illumination light and one or more wavelength bands of the light emission, where the one or more wavelength bands of the light emission define a wavelength range, and where N>1, and exposing the sample to illumination light in a background excitation band and measuring light emission from the sample in a background spectral band to obtain a background image of the sample, where the background spectral band corresponds to a wavelength within the wavelength range.

Abstract: The disclosure features methods that include exposing a biological sample to illumination light and measuring light emission from the sample to obtain N sample images, where each sample image corresponds to a different combination of a wavelength band of the illumination light and one or more wavelength bands of the light emission, where the one or more wavelength bands of the light emission define a wavelength range, and where N>1, and exposing the sample to illumination light in a background excitation band and measuring light emission from the sample in a background spectral band to obtain a background image of the sample, where the background spectral band corresponds to a wavelength within the wavelength range.

Abstract: The disclosure features methods that include exposing a biological sample to illumination light and measuring light emission from the sample to obtain N sample images, where each sample image corresponds to a different combination of a wavelength band of the illumination light and one or more wavelength bands of the light emission, where the one or more wavelength bands of the light emission define a wavelength range, and where N>1, and exposing the sample to illumination light in a background excitation band and measuring light emission from the sample in a background spectral band to obtain a background image of the sample, where the background spectral band corresponds to a wavelength within the wavelength range.

Abstract: The disclosure features methods that include exposing a biological sample to illumination light and measuring light emission from the sample to obtain N sample images, where each sample image corresponds to a different combination of a wavelength band of the illumination light and one or more wavelength bands of the light emission, where the one or more wavelength bands of the light emission define a wavelength range, and where N>1, and exposing the sample to illumination light in a background excitation band and measuring light emission from the sample in a background spectral band to obtain a background image of the sample, where the background spectral band corresponds to a wavelength within the wavelength range.

Abstract: The disclosure features methods that include exposing a biological sample to illumination light and measuring light emission from the sample to obtain N sample images, where each sample image corresponds to a different combination of a wavelength band of the illumination light and one or more wavelength bands of the light emission, where the one or more wavelength bands of the light emission define a wavelength range, and where N>1, and exposing the sample to illumination light in a background excitation band and measuring light emission from the sample in a background spectral band to obtain a background image of the sample, where the background spectral band corresponds to a wavelength within the wavelength range.