The generation of laser desorption / ionization (LDI) method can be traced back to the late 1960s.Like SIMS-MSI imaging, LDI-MSI needs to focus the laser on the sample and focus the spot to the diffraction limit to achieve the best spatial resolution.Early laser desorption / ionization mass spectrometers focused pulsed laser on thin film samples in vacuum.There are two ways to realize it: transmission focusing (laser focusing on the back of the sample) or reflection focusing (laser focusing on the front of the sample).The LDI-TOF mass spectrometer was first designed by Fenner et al. In 1966.The instrument uses a 694 nm ruby laser to focus on thin metal foil or glass to sputter materials.The focal spot size is 50μm. The resolution of the mass spectrum is 30. Because of the thermal effect of laser sputtering, the spot on the sample is enlarged.
LDI-MSI research began in the 1970s. Hillenkamp and his colleagues improved Fenner’s design, applied lasers to the field of mass spectrometry imaging, and developed Laser Microprobe Mass Spectrometer (LMMS) and Laser Microprobe Mass Analyzer (LAMMA).They doubled the frequency of a 694 nm ruby laser to generate a 347 nm ultraviolet (UV) laser, focused it on a solid sample (without matrix) on a glass slide through a lens, and the glass slide was also used as a vacuum window of a mass spectrometer.Using different mass analyzers (such as TOF and FTICR) to collect ion signals, ions with mass m / Z less than 200 Da can be detected.The system uses a lens with high numerical aperture, which is similar to the design of a microscope system. The diameter of the laser focus spot is up to 500 nm.
In the late 1970s, the design of hillenkamp et al. Was later developed into a commercial instrument – laser microprobe mass analyzer, Lamma 500 (Leybold Heraeus).Because the sample is required to be thick, then the Lamma 1000 was introduced. The light source used is the third harmonic generation (355 nm) or fourth harmonic generation (266 nm) of the 1064 nm Nd: YAG infrared laser. It is focused by a 50 times lens with a numerical aperture of 0.6, and the imaging resolution can reach 2.5μm。The emergence and development of Lamma 1000 has laid a good foundation for the development of MALDI imaging method.This method has been used in the analysis of inorganic and biological samples, such as single bacterial cell analysis, and can detect small molecules of M / Z 150.Since then, Fourier transform ion cyclotron resonance (FTICR) mass spectrometer and laser desorption ion probe instrument have been developed.The traditional reflection focusing method is used to get 5 ~ 10μIt can be used for the analysis of mouse embryonic fibroblasts and extracellular biological samples.In 1985, it was reported that LDI mass spectral imaging could obtain mass spectral imaging with a mass range of about 1000 DA and a lateral resolution of about 1μm, similar to that of TOF SIMS.
In the mid-1980s, the emergence of MALDI expanded the application of LDI in mass spectrometry.In 1997, Caprioli’s team focused a 337 NM NITROGEN LASER AT ~ 25m to analyze samples with either a matrix of 2,5-dihydroxybenzoic acid (DHB) or a cyanide-4-hydroxycinnamic acid (CHCA)2,5-dihydroxybenzoic acid, by associating Maldi mass spectrometry with sample scanning, mass spectrometry imaging of multiple biological samples has been realized. After that, the quality range of MSI detection methods based on laser becomes wider and wider. Coupled with the continuous development and improvement of mass spectrometry imaging technology and instruments, MALDI-MSI is widely used in the field of mass spectrometry imaging.At the same time, due to the development of tandem mass spectrometry and proteomics, mass spectrometry imaging has become an effective method to analyze biomolecules.It can be used to study the recognition and spatial localization of elements, metabolites, lipids, drugs, even peptides and proteins in biological tissues.The current MALDI-MSI instrument can detect the mass spectrum with molecular mass up to 30 kDa and image the mass spectrum of proteins with molecular mass more than 20 kDa (the lateral resolution is 5μm）。The lateral resolution of commercial MALDI instruments is increasing from 50μM direction 10μM continuous improvement.
With the development of ambient and atmospheric ionization methods, a variety of new MSI methods have been developed.Most of these imaging methods use pulsed laser to desorb / ionize the sample.For example, MALDI can carry out experiments under normal pressure;Matrix free MS imaging can be realized by using infrared laser or short pulse laser;New matrix materials and conditions are used to produce highly charged ions;Laser sputtering combined with ionization or chemical ionization after electrospray.These ambient / atmospheric laser ionization methods provide new ideas for direct and rapid tissue mass spectrometry imaging.
All examples of MSI methods described above are in microprobe mode, i.e. the mass spectra of each point on the sample surface are collected sequentially.Another method is microscope mode MSI. In this mode, the ions generated in the sample area and the spatial information of the corresponding collection area are detected simultaneously, as shown in Figure 2.4.The original design of microscope mode MALDI-MSI is based on TOF-SIMS mass spectrometry.The mass spectrometer was combined with nitrogen laser at 337 nm, equipped with electrostatic analyzer, dual microchannel plate, fluorescent screen detector assembly and CCD camera.The diameter of the spot focused on the sample is 200μm. The light intensity energy density is 20 MJ / cm2.Because the detector can not detect all the ions fast enough, it can only collect the ion signals of interest.When the laser irradiates the sample, the ions from the sample are amplified on the detector to realize mass spectrometry imaging.The system can get a lateral resolution of 4μM peptide and protein samples.
Using mid infrared laser in microscope mode MSI can realize mass spectrometry imaging below the diffraction limit of laser.For example, peptide imaging with a lateral resolution of 4μm can be achieved using a 2.94μm wavelength Er: Yag Laser. Microscope mode mass spectrometry imaging can be integrated with electronics-related technologies, but it needs expensive professional instruments. However, when the microscope mode is applied to SIMS, the ion beam can be focused by a method similar to focusing the beam.With the rapid development of new ionization and sampling methods, environmental LDI-MSI has gradually become a new field.This method generally uses infrared laser, because the infrared wavelength is long, which makes it at a disadvantage in the lateral resolution.However, if better optical configuration such as transmission focusing and high numerical aperture objective is adopted, the infrared laser imaging performance can be closer to the requirements of high resolution MSI.
The improvement of lateral resolution in mass spectrometry imaging experiment is realized by reducing the spot size of laser beam.The minimum spot diameter D of a focused laser beam can be expressed as
(d=λ/2nsinθ=λ/2NA (1-1) )
Among λ Is the laser wavelength, n is the refractive index of the medium andθ is the half angle of the beam leaving the lens.nsin is known as the numerical aperture (NA) of a lens. In high-quality optical systems, the number can reach about 1.5.According to the above idea, the coaxial objective lens can be used in MALDI to reduce the laser spot size to 1μm or less.However, this kind of coaxial objective lens is usually large and must work at a short distance, which requires the lens to be very close to the sample and is not suitable for ion detection after laser desorption and sputtering.It is difficult to realize the desorption and ionization of sample molecules with traditional high numerical aperture objective because the lens itself blocks the flight path of ions from the sample.This problem is usually solved by using a lens with a central hole.This kind of imaging mass spectrometer is reported in the literature. It uses a central hole objective with numerical aperture of 0.6, inner diameter of 6 mm and working distance of 16 mm.The objective is placed in a vacuum chamber above the sample, and the focused laser is guided to the sample.The desorbed and ionized ions pass through the central hole of the objective lens and accelerate into the flight tube of the mass spectrometer.The lateral resolution of the instrument can reach 0.6μm. The step size is 0.25μm。Controlling the deposition, sublimation and recrystallization of the matrix can achieve high spatial resolution, such as the lateral resolution less than 10μm. Mass spectrum imaging of protein with mass up to 27 kDa andMass spectral imaging of biological samples with lateral resolution of 2μm.
For a specific laser wavelength, the diffraction limit corresponding to this wavelength has been given by the above formula 2-1.However, in the experiment, many parameters can not meet the requirements of diffraction limit, such as desorption laser has a certain divergence angle from the laser, spherical and aspherical lens system processing and installation errors, which increase the difficulty of obtaining diffraction limit spot.In addition, in addition to optimizing the processing error and assembly of the lens surface, the focusing effect can also be optimized by beam shaping.In practical experiments, the diameter D of the focused laser beam can be expressed a D(f)=(4fλM^2)/πD(0) (1-2)
Among λ is the laser wavelength, f is the focal length of the lens, M2 is the beam quality factor, and D (0) is the unfocused laser beam diameter.With the improvement of beam quality, the beam quality factor decreases.For an ideal Gaussian beam, the beam quality factor M2 is 1.When low power CW laser works in transverse oscillation mode, M2 can be as low as 1.1, while high power multimode laser M2 can be more than 10.It can be seen from the above formula that the laser wavelength, the focal length of the lens and the M2 factor have a great influence on the final focusing of the light source.Among these three factors, the laser with shorter wavelength, especially the light source with wavelength less than 150nm, is more difficult to produce.Recently, Ilya Kuznetsov et al. Achieved a mass spectrum imaging with a transverse resolution of 75 nm by using a 46.9 nm EUV light source.
At present, the reflection focusing scheme is mostly used in mass spectrometry imaging, that is, the laser spot is focused on the front of the sample.In 1997, this reflective focusing design geometry was reported.This design is usually realized by Schwarzschild objective lens.Schwarzschild objective is a group of mirrors. A convex lens with a smaller diameter is paired with a concave auxiliary lens with a larger diameter and a central aperture.The achromatic objective has a long working distance, which can make the spatial resolution less than 10μm。So as to achieve better laser desorption ionization.For example, a FTICR mass spectrometer can obtain a spatial resolution of 1μm by focusing its laser pulses through a Schwarzschild objective.In this system, the sample surface is analyzed by desorption / ionization with 337 nm laser (nitrogen laser). For some applications, 213 nm laser is used for post ionization (5-fold frequency of Nd: YAG).Similarly, t is also possible to obtain transverse resolution less than 1μm by using the line scan mode in the Schwarzschild microscopic system.In this system, a 524 nm Nd: YLF or Nd: YAG Frequency Doubled 532 nm laser is used to desorb / ionize the sample, and a 800 nm Ti: sapphire femtosecond laser is used to post ionize the sample. The mass spectrum image of rhodamine dye deposited on the copper grid is obtained, and its transverse resolution is less than 2μm。
In LDI-MSI, besides reflective focusing scheme, transmission focusing scheme is also used.Like laser microprobe, transmission focusing needs larger numerical aperture, so it is necessary to redesign maldi-msi instrument.Thinner tissue sections must also be used because the laser must penetrate the sample to desorb the matrix and analyte on the surface of the sample.Lens focusing is different from reflection focusing, which tends to desorb and ionize larger particles, which may reduce ion efficiency.Nevertheless, the maldi-msi instrument with transmission design has been developed.A Commercial MALDI-TOF Mass Spectrometer uses a 355nm laser to image 5μm thick tissue sections and individual mammalian cells through a 100x microscope objective.In the transmission focusing mode, the minimum laser diameter corresponding to mass spectrum imaging is 1μm. By oversampling, the step size is as low as 0.5μm. For example, mass spectrum imaging of biological tissue with laser Facula of 2μm and step length of 2μm has been reported,The ND: YLF LASER is focused to 1 m and the scanning step is 2.5μm.It is worth noting that the transmission focusing scheme is not suitable for mass spectrometry imaging with VUV light source, because the interaction depth between VUV light and sample is very shallow, less than 100 nm.
MALDI-MSI is now widely used in LDI-MSI.People try to improve the quality of laser beam in MALDI system by transmitting laser through fiber, but this method is not suitable for high power pulsed laser.It is a good choice to use pinhole aperture in MALDI-MSI.A good way is to place pinholes in the path of laser propagation.Commercial MALDI-MSI instruments can perform mass spectrometry imaging with 5 m lateral resolution through 25μm ceramic pinholes.In MALDI-MSI, Gaussian beams can also be focused through pinholes and aspheric lenses to achieve high spatial resolution.In order to improve the lateral resolution of MALDI-MSI, besides optimizing the laser focusing scheme, there are also oversampling methods.
The oversampling method does not need large-scale modification of MALSI-MSI instrument, so the transverse resolution can be smaller than the focal spot diameter of laser beam.In the oversampling, the scanning step is set lower than the laser beam diameter to obtain higher spatial resolution mass spectrometry imaging.A 100μm ~ 200μm laser spot mass spectral imaging system can obtain 40μm transverse resolution by setting the step length of 25μm to scan samples. A recent paper reported that 5M resolution mass spectral imaging can be achieved by over sampling the laser beam diameter of 20.However, oversampling takes a long time to collect data, and it is easy to cause signal loss.
Another method is sample stretching.The tissue sections were deposited on glass beads embedded in paraffin film and stretched evenly in two dimensions.In order to maintain the stability of the paraffin film, the paraffin film is connected to the glass slide.The stretching method has no diffusion problem and can increase the spatial resolution and extract analytes more effectively.