Supplemental data

I. Characterization.The morphology and the selected area electron diffraction (SAED) pattern of both samples were inspected by a

transmission electron microscope (TEM, JEOL-1010, accelerating voltage 100 KV, resolution of point image 0.4

nm) with TEM films (6.5 cm×9.0 cm, China Lucky Film Co., Ltd.). After treated by ultrasonic vibration for 5 min,

powder samples were filtered twice by 0.22 μm MILLEX® GP Filter Unit. The filtered samples for TEM

investigation were prepared by dispersing nanoparticles in ethanol to obtain a suspension with the assistance of

ultrasonification, and then dropping one drop of the suspension on a copper TEM grid coated with a carbon film.

Cell samples for TEM investigation were prepared via a standardization procedure by centrifuging these cells,

fixing them with glutaraldehyde and OsO4 solutions, dehydrating them in ethanol, embedding them in epoxy resin

(Epon 812), polymerizing them in an oven, sectioning the block of epoxy polymer with LEICA ULTRACUT R to

50 nm, picking ultrathin sections off with cooper grids, and then staining the sections with lead citrate and uranyl

acetate.

The XRD patterns of powder samples were acquired with a Rigaku D/max-γB X-ray diffractometer system in a

conventional 2θ reflection geometry using Cu Kα radiation (λ=0.15418 nm).

The upconversion fluorescence spectra of powder samples were recorded by the Hitachi F-2500 fluorescence

spectrophotometer (instrument parameters: 2.5 nm for excitation slit, 2.5 nm for emission slit, 400 V for

photomultiplier tube voltage and 0.04 s for response time) with an external diode laser (980 nm, laser output

power: 0-1.3 W, continuous wave with 1 m fiber (number aperture (NA): 0.22 and Ø100 μm), Beijing Hi-Tech

Optoelectronics Co., Ltd, P R China) as the excitation source in place of the xenon lamp in the spectrometer. The

measurements were made at room temperature.

The FT-IR spectra of samples were measured with a Varian 800 FT-IR Scimitar Series using the potassium

bromide (KBr) pellet technique. In making the KBr pellets, 0.0010 g sample 1 or 2 was mixed with 0.1000 g KBr

powders. Each FT-IR spectrum was collected from 400 to 4000 cm−1. In vitro bright field images and upconversion fluorescence images of Jeko-1 and Raji cell lines, under a 980 nm

laser excitation, were performed on a Nikon Eclipse Ti-S inverted fluorescence microscope equipped with a 0-10

W adjustable CW 980 nm laser (BWT Beijing LTD, China) and an Andor iXon3 EMCCD digital camera as the

signal collector. Blue, green and red fluorescence were detected through three optical modules.

Phenotypes of Jeko-1 and Raji cell lines were analyzed by flow cytometry using the BD AccuriTm C6 flow

cytometry (BD Biosciences, CA). CD20 and CD5 expressions on Jeko-1 and Raji cell surfaces were detected using

FITC-conjugated monoclonal mouse anti-human CD20 (Clone L27, Code. No 347673, BD Biosciences, CA) and

PE-conjugated monoclonal mouse anti-human CD5 (Clone L17F12, Code. No 347307, BD Biosciences, CA).

Immunofluorescence of Jeko-1 and Raji cell lines was performed on cells spun on glass slides. These cells were

fixed in methanol/acetone 50:50 at -20℃ for 10 min, and stored at -80℃ until use. Primary CD5 antibody was

diluted in 5% goat serum and applied for 1 h at room temperature. FITC fluorescence label (diluted 1/100) was

applied for 30 min at room temperature. Primary CD20 antibody was performed with the same method as that of

CD5, but labeled with Cy3 fluorescence label. Both of them followed by staining with DAPI and observed with

the inverted fluorescence microscope.

In vivo upconversion fluorescence imaging of CD20- and CD5-nanoprobes in nude model mouse of Jeko-1 cells

was detected by Berthold LB983 NC100 (German) equipped with a 0-30 W WG1233B3-980nm CW laser system

(Beijing Energy Photoelectric Technology Co. Ltd., China).

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II. Graphics

Figure S1. XRD patterns of samples

Figure S2. The size distributions of hydrophobic sample 1 and 2 counted over 300 particles. Gaussian fits give the most probable

diameters of 57.4±0.4 and 59.7±0.4 nm with the FWHMs of 16.3 and 18.7 nm for them.

Figure S3. Upconversion luminescence spectra of samples under a 980 nm laser excitation.

Figure S4. Double logarithmic plots of the integrated upconversion luminescence intensities at different wavelengths from Er3+ and Tm3+

ions as a function of pump powers. The solid lines are of linear fit.

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Figure S5. Energy level diagram of Tm3+, Yb3+ and Er3+ ions, as well as the proposed upconversion mechanisms to produce the visible

luminescent bands with Stark splitting in NaYF4 host materials.

Figure S6. Plot of upconversion luminescence intensities as a function of exposure time

Figure S7. Schematic illustration of surface oxidation and bio-conjugation of nanoprobes. After two hydrophobic samples are

converted to be hydrophilic, CD20 and CD5 antibodies are conjugated with them via EDC/NHS agents to become CD20- and CD5-

nanoprobes in order to identify mantle cell lymphoma from B-cell lymphoma.

Figure S8. FT-IR spectra of samples before and after oxidization.

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Figure S9. MnO2 precipitation (a) and Non-MnO2 precipitation (b)

Figure S10. Upconversion luminescence spectra of the oxidized samples under a 980 nm laser excitation.

Figure S11. Bright field (a, d), blue (b) and green (e) upconversion fluorescence images, as well as single overlays (c and f). (10×, 1 bar =

(b)(a)

(c)(b)(a)

(f)(d) (e)

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50 μm). In where, quadrangles of short dash dot are the expanded beam spot of laser.

Figure S12. Bright field and blue/green upconversion fluorescence images of Jeko-1 cells, as well as their overlays (20×, 1 bar =20 μm).

Figure S13. Bright field and blue/green upconversion fluorescence images of Jeko-1 cells, as well as their overlays (40×, 1 bar =10 μm).

Figure S14. Bright field and blue/green upconversion fluorescence images of Jeko-1 cells, as well as their overlays (40×, 1 bar =10 μm).

Figure S15. Bright field and blue/green upconversion fluorescence images of Jeko-1 cells, as well as their overlays (40×, 1 bar =10 μm).

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Figure S16. Bright field and blue/green upconversion fluorescence images of Jeko-1 cells, as well as their overlays (40×, 1 bar =10 μm).

Figure S17. Bright field and blue/green upconversion fluorescence images of Jeko-1 cells, as well as their overlays (40×, 1 bar =10 μm).

Figure S18. Bright field and blue/green upconversion fluorescence images of Jeko-1 cells, as well as their overlays (40×, 1 bar =10 μm).

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Figure S19. Bright field and blue single-labeling upconversion fluorescence images of Raji cells, as well as the overlay (40×, 1 bar =10

μm)

III. The proposed upconversion mechanismsConsidering the photon energy of 980 nm laser and the energy levels of Yb3+, Er3+ and Tm3+ ions, the proposed

upconversion mechanisms to produce the visible upconversion luminescence are shown in Figure S5, in where

phonons of the host matrix are also needed to compensate the mismatches of the energy levels between Yb3+, Er3+

and Tm3+ ions with the photon energy of 980 nm laser. Here, Yb3+ ions as a sensitizer can firstly transfer the

absorbed energy from the 2F5/2 state of Yb3+ ions to the 4I11/2 state of Er3+ ions nearby in Yb3+-Er3+ co-doping

systems or the 3H5 state of Tm3+ with the help of phonons in Yb3+-Tm3+ systems, and then transfer another absorbed

energy to different energy levels of Er3+ and Tm3+ ions step by step. Thus, two-photon upconversion luminescence

of Er3+ ions at 543 and 657 nm, as well as three/four-photon upconversion luminescence of Tm3+ ions at 477 and

361 nm can be obviously obtained in Figure S3. However, a customary upconversion luminescence of Tm3+ ions at

800 nm is not observed in Figure S3. This is duo to a fact that in Hitachi F-2500 fluorescence spectrophotometer,

the quantum efficiency of photomultiplier tube (Hamamatsu R3788) at this wavelength is only 0.02%, which

is much lower than 30% at 250 nm.

IV. A simple explanation on the improved upconversionSome adhesive materials such as sodium oleate and ligands on the particles’ surfaces play a similar role to the

shell layers of the core-shell type nanoparticles in the improved upconversion fluorescence. That is to say that the

‘dormant’ lanthanide ions doped within the most outside spherical shell of nanoparticles are aroused by the ligand

fields around lanthanide ions between the most outside spherical shell and the adhesive materials. The ligand fields

split 2S+1LJ levels of these lanthanide ions into several Stark levels, and further mix their different J states to

weaken the selection rules of radiation transitions. This means that lanthanide ions on the particles’ surfaces have

been converted their roles from the ‘dormant’ state to the ‘activated’ state, and become new luminescence centers.

Thus, the enhancement of upconversion fluorescence intensity can be ascribed to the additional upconversion

fluorescence from lanthanide ions on the particles’ surfaces.

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